Protein Detection Methods and Application

Protein Analysis MethodsProtein Analysis Methods
andand ApplicationApplication
LECTURE BYLECTURE BY
ANGEL L. SALAMAN-BAYRON, Ph.D.ANGEL L. SALAMAN-BAYRON, Ph.D.

OUTLINEOUTLINE
 Gel ElectrophoresisGel Electrophoresis
 Gel Electrophoresis Under denaturingGel Electrophoresis Under denaturing
conditionsconditions
 Gel Electrophoresis Under non-denaturingGel Electrophoresis Under non-denaturing
conditionsconditions
 2D Gel Electrophoresis2D Gel Electrophoresis
 Colorimetric AssaysColorimetric Assays
 Protein ImmunostainingProtein Immunostaining
 Proteins Colorimetric AssayProteins Colorimetric Assay
 ElectrofocusingElectrofocusing
 Non-Traditional (Other) MethodsNon-Traditional (Other) Methods

Gel ElectrophoresisGel Electrophoresis
 Gel electrophoresisGel electrophoresis is a technique used for theis a technique used for the
separation of deoxyribonucleic acid (DNA),separation of deoxyribonucleic acid (DNA),
ribonucleic acid (RNA), or protein moleculesribonucleic acid (RNA), or protein molecules
using an electric current applied to a gel matrix.using an electric current applied to a gel matrix.
DNA Gel electrophoresis is generally only usedDNA Gel electrophoresis is generally only used
after amplification of DNA via PCR. It is usuallyafter amplification of DNA via PCR. It is usually
performed for analytical purposes, but may beperformed for analytical purposes, but may be
used as a preparative technique prior to use ofused as a preparative technique prior to use of
other methods such as mass spectrometry,other methods such as mass spectrometry,
PCR, cloning, DNA sequencing, Southern andPCR, cloning, DNA sequencing, Southern and
Western Blotting for further characterization.Western Blotting for further characterization.

Gel ElectrophoresisGel Electrophoresis
 Proteins, unlike nucleic acids, can have varying chargesProteins, unlike nucleic acids, can have varying charges
and complex shapes, therefore they may not migrate intoand complex shapes, therefore they may not migrate into
the polyacryl amide gel at similar rates, or at all, whenthe polyacryl amide gel at similar rates, or at all, when
placing a negative to positive EMF on the sample.placing a negative to positive EMF on the sample.
Proteins therefore, are usually denatured in the presenceProteins therefore, are usually denatured in the presence
of a detergent such as sodium dodecyl sulfate/of a detergent such as sodium dodecyl sulfate/
(SDS/SDP) that coats the proteins with a negative charge.(SDS/SDP) that coats the proteins with a negative charge.
Generally, the amount of SDS bound is relative to the sizeGenerally, the amount of SDS bound is relative to the size
of the protein (usually 1.4g SDS per gram of protein), soof the protein (usually 1.4g SDS per gram of protein), so
that the resulting denatured proteins have an overallthat the resulting denatured proteins have an overall
negative charge, and all the proteins have a similarnegative charge, and all the proteins have a similar
charge to mass ratio. Since denatured proteins act likecharge to mass ratio. Since denatured proteins act like
long rods instead of having a complex tertiary shape, thelong rods instead of having a complex tertiary shape, the
rate at which the resulting SDS coated proteins migrate inrate at which the resulting SDS coated proteins migrate in
the gel is relative only to its size and not its charge orthe gel is relative only to its size and not its charge or
shape.shape.

SDS-PAGE (PolyAcrylamide GelSDS-PAGE (PolyAcrylamide Gel
Electrophoresis)Electrophoresis)
 The purpose of this method is to separateThe purpose of this method is to separate
proteins according to their size, and noproteins according to their size, and no
other physical feature. In order toother physical feature. In order to
understand how this works, we have tounderstand how this works, we have to
understand the two halves of the name:understand the two halves of the name:
SDSSDS andand PAGEPAGE

SDS:SDS: Sodium Dodecyl SulfateSodium Dodecyl Sulfate

PAGE:PAGE: Polyacrylamide Gel ElectrophoresisPolyacrylamide Gel Electrophoresis

 SDSSDS (sodium dodecyl sulfate) is a detergent(sodium dodecyl sulfate) is a detergent
(soap) that can dissolve hydrophobic molecules(soap) that can dissolve hydrophobic molecules
but also has a negative charge (sulfbut also has a negative charge (sulfATEATE))
attached to it. Therefore, if a cell is incubatedattached to it. Therefore, if a cell is incubated
with SDS, the membranes will be dissolved andwith SDS, the membranes will be dissolved and
the proteins will be solubilized by the detergent,the proteins will be solubilized by the detergent,
plus all the proteins will be covered with manyplus all the proteins will be covered with many
negative charges. The end result has twonegative charges. The end result has two
important features:important features:

1) all proteins contain only primary structure and1) all proteins contain only primary structure and

2) all proteins have a large negative charge which2) all proteins have a large negative charge which
means they will all migrate towards the positive polemeans they will all migrate towards the positive pole
when placed in an electric fieldwhen placed in an electric field

 The top portion of the figure showsThe top portion of the figure shows
a protein with negative and positivea protein with negative and positive
charges due to the charged R-charges due to the charged R-
groups of the particular amino acidsgroups of the particular amino acids
in the protein. The large Hin the protein. The large H
represents hydrophobic domainsrepresents hydrophobic domains
where nonpolar R-groups havewhere nonpolar R-groups have
collected in an attempt to get awaycollected in an attempt to get away
from the polar water that surroundsfrom the polar water that surrounds
the protein. The bottom portionthe protein. The bottom portion
shows that SDS can break upshows that SDS can break up
hydrophobic areas and coathydrophobic areas and coat
proteins with many negativeproteins with many negative
charges which overwhelms anycharges which overwhelms any
positive charge in the protein due topositive charge in the protein due to
positively charged R-groups. Thepositively charged R-groups. The
resulting protein has beenresulting protein has been
denatured by SDS (reduced to itsdenatured by SDS (reduced to its
primary structure) and as a resultprimary structure) and as a result
has been linearized.has been linearized.

Protein Detection Methods and Application

ElectrophoresisElectrophoresis
This Figure shows a slab of polyacrylamide (dark
gray) with tunnels (different sized red rings with
shading to depict depth) exposed on the edge.
Notice that there are many different sizes of
tunnels scattered randomly throughout the gel.

Native" or "non-denaturing" gelNative" or "non-denaturing" gel
electrophoresiselectrophoresis
 ……is run in the absence of SDS. While in SDS-PAGE theis run in the absence of SDS. While in SDS-PAGE the
electrophoretic mobility of proteins depends primarily on theirelectrophoretic mobility of proteins depends primarily on their
molecular mass, in native PAGE the mobility depends on both themolecular mass, in native PAGE the mobility depends on both the
protein's charge and its hydrodynamic size.protein's charge and its hydrodynamic size.
 The electric charge driving the electrophoresis is governed by theThe electric charge driving the electrophoresis is governed by the
intrinsic charge on the protein at the pH of the running buffer. Thisintrinsic charge on the protein at the pH of the running buffer. This
charge will, of course, depend on the amino acid composition of thecharge will, of course, depend on the amino acid composition of the
protein as well as post-translational modifications such as additionprotein as well as post-translational modifications such as addition
of sialic acids.of sialic acids.
 Since the protein retains its folded conformation, its hydrodynamicSince the protein retains its folded conformation, its hydrodynamic
size and mobility on the gel will also vary with the nature of thissize and mobility on the gel will also vary with the nature of this
conformation (higher mobility for more compact conformations,conformation (higher mobility for more compact conformations,
lower for larger structures like oligomers). If native PAGE is carriedlower for larger structures like oligomers). If native PAGE is carried
out near neutral pH to avoid acid or alkaline denaturation, then itout near neutral pH to avoid acid or alkaline denaturation, then it
can be used to study conformation, self-association or aggregation,can be used to study conformation, self-association or aggregation,
and the binding of other proteins or compounds.and the binding of other proteins or compounds.

 Thus native gels can be sensitive to anyThus native gels can be sensitive to any
process that alters either the charge or theprocess that alters either the charge or the
conformation of a protein. This makesconformation of a protein. This makes
them excellent tools for detecting thingsthem excellent tools for detecting things
such as:such as:

changes in charge due to chemical degradation (changes in charge due to chemical degradation (e.g.e.g.
deamidation)deamidation)

unfolded, "molten globule", or other modifiedunfolded, "molten globule", or other modified
conformationsconformations

oligomers and aggregates (both covalent and non-oligomers and aggregates (both covalent and non-
covalent)covalent)

binding events (protein-protein or protein-ligand)binding events (protein-protein or protein-ligand)

 These properties, and their relatively high throughput,These properties, and their relatively high throughput,
make native gels excellent tools for analyzingmake native gels excellent tools for analyzing
accelerated stability samples, demonstratingaccelerated stability samples, demonstrating
comparability of different lots or processes, or examiningcomparability of different lots or processes, or examining
the effects of excipientsthe effects of excipients..
 Another advantage of native gels is that it is possible toAnother advantage of native gels is that it is possible to
recover proteins in their native state after the separation.recover proteins in their native state after the separation.
Recovery of active biological materials may, however,Recovery of active biological materials may, however,
need to be done prior to any fixing or staining.need to be done prior to any fixing or staining.
 Today many labs unfortunately ignore this valuable toolToday many labs unfortunately ignore this valuable tool
because they think native gels are just too hard to use,because they think native gels are just too hard to use,
or because they mistakenly believe they can only beor because they mistakenly believe they can only be
used with acidic proteins. Bused with acidic proteins. Bothoth basic and acidic proteins,basic and acidic proteins,
starting with commercial pre-cast gels and changing thestarting with commercial pre-cast gels and changing the
buffers can be run.buffers can be run.

It is therefore possible to screen conditions that minimize such oligomer
formation using native PAGE. In fact, the figure above shows no dimer
formation in histidine, glycine or Tris-HCl buffers (all at 20 mM),
consistent with the highly-reversible thermal unfolding of EPO in those
conditions.

2D Gel Electrophoresis2D Gel Electrophoresis
 This is a method for the separation andThis is a method for the separation and
identification of proteins in a sample byidentification of proteins in a sample by
displacement in 2 dimensions oriented atdisplacement in 2 dimensions oriented at
right angles to one another. This allowsright angles to one another. This allows
the sample to separate over a larger area,the sample to separate over a larger area,
increasing the resolution of eachincreasing the resolution of each
component. A good, general, up to datecomponent. A good, general, up to date
reference on this subject is Görg et al,reference on this subject is Görg et al,
(2000).(2000).

How is it Performed?How is it Performed?
 Isoelectric focusingIsoelectric focusing (IEF) is used in the 1st(IEF) is used in the 1st
Dimension (Righetti, P.G., 1983). This separatesDimension (Righetti, P.G., 1983). This separates
proteins by their charge (pI).proteins by their charge (pI).
 SDS-PAGESDS-PAGE in the 2nd Dimension. Thisin the 2nd Dimension. This
separates proteins by their size (molecularseparates proteins by their size (molecular
weight, MW).weight, MW).
 The procedure is known asThe procedure is known as ISO-DALTISO-DALT: iso for: iso for
isoelectric focusing and dalt for dalton weightisoelectric focusing and dalt for dalton weight

 Ispelectrofocusing (IEF)Ispelectrofocusing (IEF) pH gradients can be generatedpH gradients can be generated
by adding ampholytes to an acrylamide gel. These are aby adding ampholytes to an acrylamide gel. These are a
mixture of amphoteric species with a range of pI values.mixture of amphoteric species with a range of pI values.
They are used for carrier ampholyte IEF and in this caseThey are used for carrier ampholyte IEF and in this case
the gel may be pre-focused before sample application.the gel may be pre-focused before sample application.
 Ampholyte properties:Ampholyte properties:

Even conductivityEven conductivity

High buffering capacityHigh buffering capacity

Soluble at isoelectric pointSoluble at isoelectric point

Minimum interaction with focused proteinsMinimum interaction with focused proteins
 ImmobilinesImmobilines, s, similar to Ampholytes but have beenimilar to Ampholytes but have been
immobilised within the polyacrylamide gel producing animmobilised within the polyacrylamide gel producing an
immobilised pH gradient or IPG that does not need to beimmobilised pH gradient or IPG that does not need to be
pre-focused.pre-focused.

 ……is generally used as a component of proteomics and is the stepis generally used as a component of proteomics and is the step
used for the isolation of proteins for further characterization by massused for the isolation of proteins for further characterization by mass
spectroscopy. Purposes, firstly for thespectroscopy. Purposes, firstly for the large scale identificationlarge scale identification of allof all
proteins in a sample. This is undertaken when the global proteinproteins in a sample. This is undertaken when the global protein
expression of an organism or a tissue is being investigated and isexpression of an organism or a tissue is being investigated and is
best carried out on model organisms whose genomes have beenbest carried out on model organisms whose genomes have been
fully sequenced. In this way the individual proteins can be morefully sequenced. In this way the individual proteins can be more
readily identified from the mass spectrometry data. The second usereadily identified from the mass spectrometry data. The second use
of this technique is differential expression, this is when you compareof this technique is differential expression, this is when you compare
two or more samples to find differences in theirtwo or more samples to find differences in their protein expressionprotein expression..
For instance, you may be looking at drugs resistance in a parasite.For instance, you may be looking at drugs resistance in a parasite.
In this case you might like to compare a resistant organism to aIn this case you might like to compare a resistant organism to a
susceptible one in an attempt to find the changes responsible for thesusceptible one in an attempt to find the changes responsible for the
resistance. Here the sequence requirements of the organism are notresistance. Here the sequence requirements of the organism are not
as important, as you are looking for a relatively small number ofas important, as you are looking for a relatively small number of
differences and so can devote more time to the identification of eachdifferences and so can devote more time to the identification of each
protein.protein.

Proteins Colorimetric AssayProteins Colorimetric Assay
 Hartree-Lowry and Modified Lowry Protein Assays:Hartree-Lowry and Modified Lowry Protein Assays: The Lowry assayThe Lowry assay
(1951) is an often-cited general use protein assay. For some time it(1951) is an often-cited general use protein assay. For some time it
was the method of choice for accurate protein determination for cellwas the method of choice for accurate protein determination for cell
fractions, chromatography fractions, enzyme preparations, and sofractions, chromatography fractions, enzyme preparations, and so
on. The bicinchoninic acid (BCA) assay is based on the sameon. The bicinchoninic acid (BCA) assay is based on the same
principle and can be done in one step, therefore it has beenprinciple and can be done in one step, therefore it has been
suggested (Stoscheck, 1990) that the 2-step Lowry method issuggested (Stoscheck, 1990) that the 2-step Lowry method is
outdated. However, the modified Lowry is done entirely at roomoutdated. However, the modified Lowry is done entirely at room
temperature. The Hartree version of the Lowry assay, a more recenttemperature. The Hartree version of the Lowry assay, a more recent
modification that uses fewer reagents, improves the sensitivity withmodification that uses fewer reagents, improves the sensitivity with
some proteins, is less likely to be incompatible with some saltsome proteins, is less likely to be incompatible with some salt
solutions, provides a more linear response, and is less likely tosolutions, provides a more linear response, and is less likely to
become saturated.become saturated.
 Reaction Principle:Reaction Principle: Under alkaline conditions the divalent copper ionUnder alkaline conditions the divalent copper ion
forms a complex with peptide bonds in which it is reduced to aforms a complex with peptide bonds in which it is reduced to a
monovalent ion. Monovalent copper ion and the radical groups ofmonovalent ion. Monovalent copper ion and the radical groups of
tyrosine, tryptophan, and cysteine react with Folin reagent totyrosine, tryptophan, and cysteine react with Folin reagent to
produce an unstable product that becomes reduced toproduce an unstable product that becomes reduced to
molybdenum/tungsten bluemolybdenum/tungsten blue

 Biuret Protein Assay:Biuret Protein Assay: The principle of the Biuret assayThe principle of the Biuret assay
is similar to that of the Lowry, however it involves ais similar to that of the Lowry, however it involves a
single incubation of 20 min. There are very fewsingle incubation of 20 min. There are very few
interfering agents (ammonium salts being one suchinterfering agents (ammonium salts being one such
agent), and Layne (1957) reported fewer deviations thanagent), and Layne (1957) reported fewer deviations than
with the Lowry or ultraviolet absorption methods.with the Lowry or ultraviolet absorption methods.
However, the biuret assay consumes much moreHowever, the biuret assay consumes much more
material. The biuret is a good general protein assay formaterial. The biuret is a good general protein assay for
batches of material for which yield is not a problem. Thebatches of material for which yield is not a problem. The
Bradford assay is faster and more sensitive.Bradford assay is faster and more sensitive.
 Reaction Principle:Reaction Principle: Under alkaline conditionsUnder alkaline conditions
substances containing two or more peptide bonds form asubstances containing two or more peptide bonds form a
purple complex with copper salts in the reagentpurple complex with copper salts in the reagent

 Bradford protein assay:Bradford protein assay: The Bradford assay is very fast and uses about theThe Bradford assay is very fast and uses about the
same amount of protein as the Lowry assay. It is fairly accurate and samplessame amount of protein as the Lowry assay. It is fairly accurate and samples
that are out of range can be retested within minutes. The Bradford isthat are out of range can be retested within minutes. The Bradford is
recommended for general use, especially for determining protein content ofrecommended for general use, especially for determining protein content of
cell fractions and assessing protein concentrations for gel electrophoresis.cell fractions and assessing protein concentrations for gel electrophoresis.
 Assay materials including color reagent, protein standard, and instructionAssay materials including color reagent, protein standard, and instruction
booklet are available from Bio-Rad Corporation. The method described belowbooklet are available from Bio-Rad Corporation. The method described below
is for a 100 µl sample volume using 5 ml color reagent. It is sensitive to aboutis for a 100 µl sample volume using 5 ml color reagent. It is sensitive to about
5 to 200 micrograms protein, depending on the dye quality. In assays using 55 to 200 micrograms protein, depending on the dye quality. In assays using 5
ml color reagent prepared in lab, the sensitive range is closer to 5 to 100 µgml color reagent prepared in lab, the sensitive range is closer to 5 to 100 µg
protein. Scale down the volume for the "microassay procedure," which uses 1protein. Scale down the volume for the "microassay procedure," which uses 1
ml cuvettes. Protocols, including use of microtiter plates are described in theml cuvettes. Protocols, including use of microtiter plates are described in the
flyer that comes with the Bio-Rad kit.flyer that comes with the Bio-Rad kit.
 Reaction Principle:Reaction Principle: The assay is based on the observation that theThe assay is based on the observation that the
absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-
250 shifts from 465 nm to 595 nm when binding to protein occurs. Both250 shifts from 465 nm to 595 nm when binding to protein occurs. Both
hydrophobic and ionic interactions stabilize the anionic form of the dye,hydrophobic and ionic interactions stabilize the anionic form of the dye,
causing a visible color change. The assay is useful since the extinctioncausing a visible color change. The assay is useful since the extinction
coefficient of a dye-albumin complex solution is constant over a 10-foldcoefficient of a dye-albumin complex solution is constant over a 10-fold
concentration range.concentration range.

 Bicinchoninic Acid (BCA) Protein Assay (Smith):Bicinchoninic Acid (BCA) Protein Assay (Smith): TheThe
bicinchoninic acid (BCA) assay is available in kit form from Piercebicinchoninic acid (BCA) assay is available in kit form from Pierce
(Rockford, Ill.). This procedure is very applicable to microtiter plate(Rockford, Ill.). This procedure is very applicable to microtiter plate
methods. The BCA is used for the same reasons the Lowry is used.methods. The BCA is used for the same reasons the Lowry is used.
Stoscheck (1990) has suggested that the BCA assay will replaceStoscheck (1990) has suggested that the BCA assay will replace
the Lowry because it requires a single step, and the color reagent isthe Lowry because it requires a single step, and the color reagent is
stable under alkaline conditions.stable under alkaline conditions.

Both a standard assay for concentrated proteins and a micro assay forBoth a standard assay for concentrated proteins and a micro assay for
dilute protein solutions are described below.dilute protein solutions are described below.
 Reaction Principle:Reaction Principle: BCA serves the purpose of the Folin reagent inBCA serves the purpose of the Folin reagent in
the Lowry assay, namely to react with complexes between copperthe Lowry assay, namely to react with complexes between copper
ions and peptide bonds to produce a purple end product. Theions and peptide bonds to produce a purple end product. The
advantage of BCA is that the reagent is fairly stable under alkalineadvantage of BCA is that the reagent is fairly stable under alkaline
conditions, and can be included in the copper solution to allow a oneconditions, and can be included in the copper solution to allow a one
step procedure. A molybdenum/tungsten blue product is producedstep procedure. A molybdenum/tungsten blue product is produced
as with the Lowry.as with the Lowry.

Protein ImmunostainingProtein Immunostaining
 ……is a general term in biochemistry that appliesis a general term in biochemistry that applies
to any use of an antibody-based method toto any use of an antibody-based method to
detect a specific protein in a sample. The termdetect a specific protein in a sample. The term
immunostaining was originally used to refer toimmunostaining was originally used to refer to
the immunohistochemical staining of tissuethe immunohistochemical staining of tissue
sections, as first described by Albert Coons insections, as first described by Albert Coons in
1941.Now however, immunostaining1941.Now however, immunostaining
encompasses a broad range of techniques usedencompasses a broad range of techniques used
in histology, cell biology, and molecular biologyin histology, cell biology, and molecular biology
that utilize antibody-based staining methods.that utilize antibody-based staining methods.

 ImmunohistochemistryImmunohistochemistry or IHC staining of tissue sectionsor IHC staining of tissue sections
(or immunocytochemistry, which is the staining of cells),(or immunocytochemistry, which is the staining of cells),
is perhaps the most commonly applied immunostainingis perhaps the most commonly applied immunostaining
technique. While the first cases of IHC staining usedtechnique. While the first cases of IHC staining used
fluorescent dyes, other non-fluorescent methods usingfluorescent dyes, other non-fluorescent methods using
enzymes such as peroxidase and alkaline phosphataseenzymes such as peroxidase and alkaline phosphatase
are now used. These enzymes are capable of catalysingare now used. These enzymes are capable of catalysing
reactions that give a colored product that is easilyreactions that give a colored product that is easily
detectable by light microscopy. Alternatively, radioactivedetectable by light microscopy. Alternatively, radioactive
elements can be used as labels, and theelements can be used as labels, and the
immunoreaction can be visualized by autoradiography.immunoreaction can be visualized by autoradiography.

 Tissue preparation orTissue preparation or fixationfixation is essential for the preservation of cellis essential for the preservation of cell
morphology and tissue architecture.morphology and tissue architecture.

Inappropriate or prolonged fixation may significantly diminish the antibodyInappropriate or prolonged fixation may significantly diminish the antibody
binding capability.binding capability.
 Many antigens can be successfully demonstrated in formalin-fixedMany antigens can be successfully demonstrated in formalin-fixed
paraffin-embedded tissue sections. However, some antigens will notparaffin-embedded tissue sections. However, some antigens will not
survive even moderate amounts of aldehyde fixation. Under thesesurvive even moderate amounts of aldehyde fixation. Under these
conditions, tissues should be rapidly fresh frozen in liquid nitrogenconditions, tissues should be rapidly fresh frozen in liquid nitrogen
and cut with a cryostat.and cut with a cryostat.

The disadvantages of frozen sections include poor morphology, poorThe disadvantages of frozen sections include poor morphology, poor
resolution at higher magnifications, difficulty in cutting over paraffinresolution at higher magnifications, difficulty in cutting over paraffin
sections, and the need for frozen storage.sections, and the need for frozen storage.
 Alternatively, vibratome sections do not require the tissue to beAlternatively, vibratome sections do not require the tissue to be
processed through organic solvents or high heat, which can destroyprocessed through organic solvents or high heat, which can destroy
the antigenicity, or disrupted by freeze thawing.the antigenicity, or disrupted by freeze thawing.

The disadvantage of vibratome sections is that the sectioning process isThe disadvantage of vibratome sections is that the sectioning process is
slow and difficult with soft and poorly fixed tissues, and that chatter marksslow and difficult with soft and poorly fixed tissues, and that chatter marks
or vibratome lines are often apparent in the sections.or vibratome lines are often apparent in the sections.

 The detection of many antigens can be dramatically improved byThe detection of many antigens can be dramatically improved by
antigen retrievalantigen retrieval methods that act by breaking some of the proteinmethods that act by breaking some of the protein
cross-links formed by fixation to uncover hidden antigenic sites. Thiscross-links formed by fixation to uncover hidden antigenic sites. This
can be accomplished by heating for varying lengths of times (heatcan be accomplished by heating for varying lengths of times (heat
induced epitope retrieval or HIER) or using enzyme digestioninduced epitope retrieval or HIER) or using enzyme digestion
(proteolytic induced epitope retrieval or PIER).(proteolytic induced epitope retrieval or PIER).
 One of the main difficulties with IHC staining is overcoming specificOne of the main difficulties with IHC staining is overcoming specific
or non-specific background. Optimisation of fixation methods andor non-specific background. Optimisation of fixation methods and
times, pre-treatment with blocking agents, incubating antibodies withtimes, pre-treatment with blocking agents, incubating antibodies with
high salt, and optimising post-antibody wash buffers and wash timeshigh salt, and optimising post-antibody wash buffers and wash times
are all important for obtaining high quality immunostaining. Inare all important for obtaining high quality immunostaining. In
addition, the presence of positive and negative controls for stainingaddition, the presence of positive and negative controls for staining
are essential for determining specificity.are essential for determining specificity.

 AA flow cytometerflow cytometer is a technology that allows a single cell to beis a technology that allows a single cell to be
measured for a variety of characteristics, determined by looking atmeasured for a variety of characteristics, determined by looking at
how they flow in liquid. Instruments used for this can gatherhow they flow in liquid. Instruments used for this can gather
information about cells by measuring visible and fluorescent lightinformation about cells by measuring visible and fluorescent light
emissions, allowing cell sorting based on physical, biochemical andemissions, allowing cell sorting based on physical, biochemical and
antigenic traits.antigenic traits.
 It can be used for the direct analysis of cells expressing one or moreIt can be used for the direct analysis of cells expressing one or more
specific proteins. Cells are immunostained in solution usingspecific proteins. Cells are immunostained in solution using
methods similar to used for immunofluorescence, and then analyzedmethods similar to used for immunofluorescence, and then analyzed
by flow cytometry.by flow cytometry.
 Flow cytometry has several advantages over IHC including: theFlow cytometry has several advantages over IHC including: the
ability to define distinct cell populations are defined by their size andability to define distinct cell populations are defined by their size and
granularity; the capacity to gate out dead cells; improved sensitivity;granularity; the capacity to gate out dead cells; improved sensitivity;
and multi-colour analysis to measure several antigensand multi-colour analysis to measure several antigens
simultaneously. However, flow cytometry can be less effective atsimultaneously. However, flow cytometry can be less effective at
detecting extremely rare cell populations, and there is a loss ofdetecting extremely rare cell populations, and there is a loss of
architectural relationships in the absence of a tissue section.architectural relationships in the absence of a tissue section.

 Western blottingWestern blotting allows the detection of specific proteinsallows the detection of specific proteins
from extracts made from cells or tissues, before or afterfrom extracts made from cells or tissues, before or after
any purification steps. Proteins are generally separatedany purification steps. Proteins are generally separated
by size using gel electrophoresis before being transferredby size using gel electrophoresis before being transferred
to a synthetic membrane via dry, semi-dry, or wet blottingto a synthetic membrane via dry, semi-dry, or wet blotting
methods. The membrane can then be probed usingmethods. The membrane can then be probed using
antibodies using methods similar toantibodies using methods similar to
immunohistochemistry, but without a need for fixation.immunohistochemistry, but without a need for fixation.
Detection is typically performed using peroxidase linkedDetection is typically performed using peroxidase linked
antibodies to catalyse a chemiluminescent reaction.antibodies to catalyse a chemiluminescent reaction.
 Western blotting is a routine molecular biology methodWestern blotting is a routine molecular biology method
that can be used to semi-quantitatively compare proteinthat can be used to semi-quantitatively compare protein
levels between extracts. The size separation prior tolevels between extracts. The size separation prior to
blotting allows the protein molecular weight to be gaugedblotting allows the protein molecular weight to be gauged
as compared with known molecular weight markersas compared with known molecular weight markers

No bands present Neg
Bands at either p31 OR p24
AND bands present at
either gp160 OR gp120
Pos
Bands present, but pattern
does not meet criteria for
positivity
+/-
Western BlottingWestern Blotting

 Western blotting transferWestern blotting transfer
apparatusapparatus.. Schematic showingSchematic showing
the assembly of a typicalthe assembly of a typical
Western blot apparatus with theWestern blot apparatus with the
position of the position of theposition of the position of the
gel, transfer membrane andgel, transfer membrane and
direction of protein in relation todirection of protein in relation to
the electrode position. Althoughthe electrode position. Although
the image depicted here isthe image depicted here is
representative of a vertical "wet"representative of a vertical "wet"
transfer apparatus, thetransfer apparatus, the
orientation is applicable fororientation is applicable for
horizontally positioned semi-dryhorizontally positioned semi-dry
transfer apparatus.transfer apparatus.

 The enzyme-linked immunosorbent assay or ELISA is aThe enzyme-linked immunosorbent assay or ELISA is a
diagnostic method for quantitatively or semi-diagnostic method for quantitatively or semi-
quantitatively determining protein concentrations fromquantitatively determining protein concentrations from
blood plasma, serum or cell/tissue extracts in a multi-wellblood plasma, serum or cell/tissue extracts in a multi-well
plate format (usually 96-wells per plate). Broadly,plate format (usually 96-wells per plate). Broadly,
proteins in solution are adsorbed to ELISA plates.proteins in solution are adsorbed to ELISA plates.
Antibodies specific for the protein of interest are used toAntibodies specific for the protein of interest are used to
probe the plate. Background is minimized by optimizingprobe the plate. Background is minimized by optimizing
blocking and washing methods (as for IHC), andblocking and washing methods (as for IHC), and
specificity is ensured via the presence of positive andspecificity is ensured via the presence of positive and
negative controls. Detection methods are usuallynegative controls. Detection methods are usually
colorimetric or chemiluminescence based.colorimetric or chemiluminescence based.


Advantages
•Quicker since only one antibody is used
•No concern for cross-reactivity of a secondary antibody
•Double possible with different labels on primary antibodies
Disadvantages
•Labeling may reduce immunoreactivity of primary antibody
•Labeled primary antibodies are expensive
•Low flexibility in choice of primary antibody label
•Little signal amplification

Advantages
•Secondary antibody can amplify signal
•A variety of labeled secondary antibodies are available
•One secondary may be used with many primary antibodies
•Labeling does not affect primary antibody immunoreactivity
•Changing secondary allows change of detection method
Disadvantages
•Secondary antibodies may produce nonspecific staining
•Additional steps required compared to the direct method

Electron microscopyElectron microscopy
 ……or EM can be used to study the detailedor EM can be used to study the detailed
microarchitecture of tissues or cells. Immuno-microarchitecture of tissues or cells. Immuno-
EM allows the detection of specific proteins inEM allows the detection of specific proteins in
ultrathin tissue sections. Antibodies labelled withultrathin tissue sections. Antibodies labelled with
heavy metal particles (e.g. gold) can be directlyheavy metal particles (e.g. gold) can be directly
visualised using transmission electronvisualised using transmission electron
microscopy. While powerful in detecting the sub-microscopy. While powerful in detecting the sub-
cellular localization of a protein, immuno-EM cancellular localization of a protein, immuno-EM can
be technically challenging, expensive, andbe technically challenging, expensive, and
require rigorous optimization of tissue fixationrequire rigorous optimization of tissue fixation
and processing methods.and processing methods.

Protein Assays byProtein Assays by
SpectrophotometrySpectrophotometry
PrinciplesPrinciples
 A spectrophotometer consists of two instruments, namely aA spectrophotometer consists of two instruments, namely a
spectrometerspectrometer for producing light of any selected color (wavelength),for producing light of any selected color (wavelength),
and aand a photometerphotometer for measuring the intensity of light. Thefor measuring the intensity of light. The
instruments are arranged so that liquid in a cuvette can be placedinstruments are arranged so that liquid in a cuvette can be placed
between the spectrometer beam and the photometer. The amount ofbetween the spectrometer beam and the photometer. The amount of
light passing through the tube is measured by the photometer. Thelight passing through the tube is measured by the photometer. The
photometer delivers a voltage signal to a display device, normally aphotometer delivers a voltage signal to a display device, normally a
galvanometer. The signal changes as the amount of light absorbedgalvanometer. The signal changes as the amount of light absorbed
by the liquid changes.by the liquid changes.
 If development of color is linked to the concentration of a substanceIf development of color is linked to the concentration of a substance
in solution then that concentration can be measured by determiningin solution then that concentration can be measured by determining
the extent of absorption of light at the appropriate wavelength. Forthe extent of absorption of light at the appropriate wavelength. For
example hemoglobin appears red because the hemoglobin absorbsexample hemoglobin appears red because the hemoglobin absorbs
blue and green light rays much more effectively than red. Theblue and green light rays much more effectively than red. The
degree of absorbance of blue or green light is proportional to thedegree of absorbance of blue or green light is proportional to the
concentration of hemoglobin.concentration of hemoglobin.

Principles (cont.)Principles (cont.)
 When monochromatic light (light of a specific wavelength) passes through a solutionWhen monochromatic light (light of a specific wavelength) passes through a solution
there is usually a quantitative relationship (Beer's law) between the solutethere is usually a quantitative relationship (Beer's law) between the solute
concentration and the intensity of the transmitted light, that is,concentration and the intensity of the transmitted light, that is,
I = II = Ioo X 10X 10--kc1kc1
 wherewhere II sub 0 is the intensity of transmitted light using the pure solvent,sub 0 is the intensity of transmitted light using the pure solvent, II is theis the
intensity of the transmitted light when the colored compound is added, c isintensity of the transmitted light when the colored compound is added, c is
concentration of the colored compound,concentration of the colored compound, ll is the distance the light passes through theis the distance the light passes through the
solution, and k is a constant. If the light pathsolution, and k is a constant. If the light path ll is a constant, as is the case with ais a constant, as is the case with a
spectrophotometer, Beer's law may be written,spectrophotometer, Beer's law may be written,
I / II / Ioo = 10= 10--kckc
= T= T
 wherewhere kk is a new constant andis a new constant and TT is the transmittance of the solution. There is ais the transmittance of the solution. There is a
logarithmic relationship between transmittance and the concentration of the coloredlogarithmic relationship between transmittance and the concentration of the colored
compound. Thus,compound. Thus,
-log T-log T == log 1/Tlog 1/T == kckc = Optical Density (OD)= Optical Density (OD)
 The O.D. is directly proportional to the concentration of the colored compound. MostThe O.D. is directly proportional to the concentration of the colored compound. Most
spectrophotometers have a scale that reads both in O.D. (absorbance) units, which isspectrophotometers have a scale that reads both in O.D. (absorbance) units, which is
a logarithmic scale, and in % transmittance, which is an arithmetic scale. Asa logarithmic scale, and in % transmittance, which is an arithmetic scale. As
suggested by the above relationships, the absorbance scale is the most useful forsuggested by the above relationships, the absorbance scale is the most useful for
colorimetric assayscolorimetric assays

 Proteins in solution absorb ultraviolet lightProteins in solution absorb ultraviolet light
with absorbance maxima @ 280 and 200with absorbance maxima @ 280 and 200
nm. Amino acids with aromatic rings arenm. Amino acids with aromatic rings are
the primary reason for the absorbancethe primary reason for the absorbance
peak at 280 nm. Peptide bonds arepeak at 280 nm. Peptide bonds are
primarily responsible for the peak at 200primarily responsible for the peak at 200
nm. Secondary, tertiary, and quaternarynm. Secondary, tertiary, and quaternary
structure all affect absorbance, thereforestructure all affect absorbance, therefore
factors such as pH, ionic strength, etc. canfactors such as pH, ionic strength, etc. can
alter the absorbance spectrum.alter the absorbance spectrum.

 Unknown proteins or protein mixturesUnknown proteins or protein mixtures.. Use the followingUse the following
formula to roughly estimate protein concentration. Pathformula to roughly estimate protein concentration. Path
length for most spectrometers is 1 cm.length for most spectrometers is 1 cm.

ConcentrationConcentration (mg/ml) = Absorbance at 280 nm divided by path(mg/ml) = Absorbance at 280 nm divided by path
length (cm.)length (cm.)
 Pure protein of known absorbance coefficientPure protein of known absorbance coefficient.. Use theUse the
following formula for a path length of 1 cm. Concentrationfollowing formula for a path length of 1 cm. Concentration
is in mg/ml, %, or molarity depending on which typeis in mg/ml, %, or molarity depending on which type
coefficient is used.coefficient is used.

ConcentrationConcentration = Absorbance at 280 nm divided by absorbance= Absorbance at 280 nm divided by absorbance
coefficientcoefficient

To convert units, use these relationships:To convert units, use these relationships:

Mg protein/ml = % protein divided by 10 = molarity divided byMg protein/ml = % protein divided by 10 = molarity divided by
protein molecular weightprotein molecular weight
 Unknowns with possible nucleic acid contaminationUnknowns with possible nucleic acid contamination.. UseUse
the following formula to estimate protein concentration:the following formula to estimate protein concentration:

Concentration (mg/ml) = (1.55 x A280) - 0.76 x A260)Concentration (mg/ml) = (1.55 x A280) - 0.76 x A260)

Enzyme AssaysEnzyme Assays
 ……are laboratory methods for measuring enzymatic activity. They are vital forare laboratory methods for measuring enzymatic activity. They are vital for
the study of enzyme kinetics and enzyme inhibition.the study of enzyme kinetics and enzyme inhibition.
 Amounts of enzymes can either be expressed as molar amounts, as with anyAmounts of enzymes can either be expressed as molar amounts, as with any
other chemical, or measured in terms of activity, in enzyme units.other chemical, or measured in terms of activity, in enzyme units.
 Enzyme activityEnzyme activity = moles of substrate converted per unit time = rate ×= moles of substrate converted per unit time = rate ×
reaction volume. Enzyme activity is a measure of the quantity of activereaction volume. Enzyme activity is a measure of the quantity of active
enzyme present and is thus dependent on conditions,enzyme present and is thus dependent on conditions, which should bewhich should be
specifiedspecified. The SI unit is the katal, 1 katal = 1 mol s-1, but this is an. The SI unit is the katal, 1 katal = 1 mol s-1, but this is an
excessively large unit. A more practical and commonly-used value is 1excessively large unit. A more practical and commonly-used value is 1
enzyme unit (EU) = 1 μmol min-1 (μ = micro, x 10-6). 1 U corresponds toenzyme unit (EU) = 1 μmol min-1 (μ = micro, x 10-6). 1 U corresponds to
16.67 nanokatals.16.67 nanokatals.
 Specific activitySpecific activity of an enzyme is another common unit. This is the activity ofof an enzyme is another common unit. This is the activity of
an enzyme per milligram of total protein (expressed in μmol min-1mg-1).an enzyme per milligram of total protein (expressed in μmol min-1mg-1).
Specific activity gives a measurement of the purity of the enzyme. It is theSpecific activity gives a measurement of the purity of the enzyme. It is the
amount of product formed by an enzyme in a given amount of time underamount of product formed by an enzyme in a given amount of time under
given conditions per milligram of enzyme. Specific activity is equal to the rategiven conditions per milligram of enzyme. Specific activity is equal to the rate
of reaction multiplied by the volume of reaction divided by the mass ofof reaction multiplied by the volume of reaction divided by the mass of
enzyme. The SI unit is katal kg-1, but a more practical unit is μmol mg-1 min-enzyme. The SI unit is katal kg-1, but a more practical unit is μmol mg-1 min-
1. Specific activity is a measure of enzyme processivity, usually constant for1. Specific activity is a measure of enzyme processivity, usually constant for
a pure enzyme.a pure enzyme.

Enzyme AssaysEnzyme Assays
Related terminologyRelated terminology
 TheThe rate of a reactionrate of a reaction is the concentration ofis the concentration of
substrate disappearing (or product produced)substrate disappearing (or product produced)
per unit time (per unit time (molmol LL − 1− 1ss − 1)− 1)
 TheThe % purity% purity is 100% × (specific activity ofis 100% × (specific activity of
enzyme sample / specific activity of pureenzyme sample / specific activity of pure
enzyme). The impure sample has lower specificenzyme). The impure sample has lower specific
activity because some of the mass is not actuallyactivity because some of the mass is not actually
enzyme. If the specific activity of 100% pureenzyme. If the specific activity of 100% pure
enzyme is known, then an impure sample willenzyme is known, then an impure sample will
have a lower specific activity, allowing purity tohave a lower specific activity, allowing purity to
be calculated.be calculated.

Types of Enzyme AssaysTypes of Enzyme Assays
 All enzyme assays measure either the consumption of substrate or production ofAll enzyme assays measure either the consumption of substrate or production of
product over time. A large number of different methods of measuring theproduct over time. A large number of different methods of measuring the
concentrations of substrates and products exist and many enzymes can be assayed inconcentrations of substrates and products exist and many enzymes can be assayed in
several different ways. Biochemists usually study enzyme-catalyzed reactions usingseveral different ways. Biochemists usually study enzyme-catalyzed reactions using
four types of experiments:four types of experiments:
 Initial rate experimentsInitial rate experiments. When an enzyme is mixed with a large excess of the. When an enzyme is mixed with a large excess of the
substrate, the enzyme-substrate intermediate builds up in a fast initial transient. Thensubstrate, the enzyme-substrate intermediate builds up in a fast initial transient. Then
the reaction achieves a steady-state kinetics in which enzyme substrate intermediatesthe reaction achieves a steady-state kinetics in which enzyme substrate intermediates
remains approximately constant over time and the reaction rate changes relativelyremains approximately constant over time and the reaction rate changes relatively
slowly. Rates are measured for a short period after the attainment of the quasi-steadyslowly. Rates are measured for a short period after the attainment of the quasi-steady
state, typically by monitoring the accumulation of product with time. Because thestate, typically by monitoring the accumulation of product with time. Because the
measurements are carried out for a very short period and because of the large excessmeasurements are carried out for a very short period and because of the large excess
of substrate, the approximation free substrate is approximately equal to the initialof substrate, the approximation free substrate is approximately equal to the initial
substrate can be made. The initial rate experiment is the simplest to perform andsubstrate can be made. The initial rate experiment is the simplest to perform and
analyze, being relatively free from complications such as back-reaction and enzymeanalyze, being relatively free from complications such as back-reaction and enzyme
degradation. It is therefore by far the most commonly used type of experiment indegradation. It is therefore by far the most commonly used type of experiment in
enzyme kinetics.enzyme kinetics.
 Progress curve experimentsProgress curve experiments.. In these experiments, the kinetic parameters areIn these experiments, the kinetic parameters are
determined from expressions for the species concentrations as a function of time. Thedetermined from expressions for the species concentrations as a function of time. The
concentration of the substrate or product is recorded in time after the initial fastconcentration of the substrate or product is recorded in time after the initial fast
transient and for a sufficiently long period to allow the reaction to approach equilibrium.transient and for a sufficiently long period to allow the reaction to approach equilibrium.
We note in passing that, while they are less common now, progress curve experimentsWe note in passing that, while they are less common now, progress curve experiments
were widely used in the early period of enzyme kinetics.were widely used in the early period of enzyme kinetics.

Types of Enzyme Assays (cont.)Types of Enzyme Assays (cont.)
 Transient kinetics experimentsTransient kinetics experiments. In these experiments, reaction behaviour. In these experiments, reaction behaviour
is tracked during the initial fast transient as the intermediate reaches theis tracked during the initial fast transient as the intermediate reaches the
steady-state kinetics period. These experiments are more difficult to performsteady-state kinetics period. These experiments are more difficult to perform
than either of the above two classes because they require rapid mixing andthan either of the above two classes because they require rapid mixing and
observation techniques.observation techniques.
 Relaxation experimentsRelaxation experiments. In these experiments, an equilibrium mixture of. In these experiments, an equilibrium mixture of
enzyme, substrate and product is perturbed, for instance by a temperature,enzyme, substrate and product is perturbed, for instance by a temperature,
pressure or pH jump, and the return to equilibrium is monitored. The analysispressure or pH jump, and the return to equilibrium is monitored. The analysis
of these experiments requires consideration of the fully reversible reaction.of these experiments requires consideration of the fully reversible reaction.
Moreover, relaxation experiments are relatively insensitive to mechanisticMoreover, relaxation experiments are relatively insensitive to mechanistic
details and are thus not typically used for mechanism identification, althoughdetails and are thus not typically used for mechanism identification, although
they can be under appropriate conditions.they can be under appropriate conditions.
 Enzyme assays can be split into two groups according to their samplingEnzyme assays can be split into two groups according to their sampling
method:method:

Continues assaysContinues assays where the assay gives a continuous reading of activity, andwhere the assay gives a continuous reading of activity, and

Discontinuous assaysDiscontinuous assays when samples are taken from an enzyme reaction atwhen samples are taken from an enzyme reaction at
intervals and the amount of product production or substrate consumption isintervals and the amount of product production or substrate consumption is
measured in these samples. Continuous assays are most convenient, with onemeasured in these samples. Continuous assays are most convenient, with one
assay giving the rate of reaction with no further work necessary.assay giving the rate of reaction with no further work necessary.

Types of Enzyme Assays (cont.)Types of Enzyme Assays (cont.)
Direct versus coupled assaysDirect versus coupled assays
 Coupled assay for hexokinase using glucose-6-Coupled assay for hexokinase using glucose-6-
phosphate dehydrogenase.phosphate dehydrogenase.
 Even when the enzyme reaction does not result in aEven when the enzyme reaction does not result in a
change in the absorbance of light, it can still be possiblechange in the absorbance of light, it can still be possible
to use a spectrophotometric assay for the enzyme byto use a spectrophotometric assay for the enzyme by
using ausing a coupled assaycoupled assay. Here, the product of one. Here, the product of one
reaction is used as the substrate of another, easily-reaction is used as the substrate of another, easily-
detectable reaction. For example, figure 1 shows thedetectable reaction. For example, figure 1 shows the
coupled assay for the enzyme hexokinase, which can becoupled assay for the enzyme hexokinase, which can be
assayed by coupling its production of glucose-6-assayed by coupling its production of glucose-6-
phosphate to NADPH production, using glucose-6-phosphate to NADPH production, using glucose-6-
phosphate dehydrogenase.phosphate dehydrogenase.

Types of Continuous EnzymeTypes of Continuous Enzyme
AssaysAssays
 FluorometricFluorometric ((Fluorescence) is when a molecule emitsFluorescence) is when a molecule emits
light of one wavelength after absorbing light of a differentlight of one wavelength after absorbing light of a different
wavelength. Fluorometric assays use a difference in thewavelength. Fluorometric assays use a difference in the
fluorescence of substrate from product to measure thefluorescence of substrate from product to measure the
enzyme reaction. These assays are in general muchenzyme reaction. These assays are in general much
more sensitive than spectrophotometric assays, but canmore sensitive than spectrophotometric assays, but can
suffer from interference caused by impurities and thesuffer from interference caused by impurities and the
instability of many fluorescent compounds when exposedinstability of many fluorescent compounds when exposed
to light.to light.

An example of these assays is again the use of the nucleotideAn example of these assays is again the use of the nucleotide
coenzymes NADH and NADPH. Here, the reduced forms arecoenzymes NADH and NADPH. Here, the reduced forms are
fluorescent and the oxidised forms non-fluorescent. Oxidationfluorescent and the oxidised forms non-fluorescent. Oxidation
reactions can therefore be followed by a decrease inreactions can therefore be followed by a decrease in
fluorescence and reduction reactions by an increase. Syntheticfluorescence and reduction reactions by an increase. Synthetic
substrates that release a fluorescent dye in an enzyme-catalyzedsubstrates that release a fluorescent dye in an enzyme-catalyzed
reaction are also available, such as 4-methylumbelliferyl-β-D-reaction are also available, such as 4-methylumbelliferyl-β-D-
galactoside for assaying β-galactosidase.galactoside for assaying β-galactosidase.

AssaysAssays
 CalorimetricCalorimetric ((Calorimetry) is the measurement of the heat releasedCalorimetry) is the measurement of the heat released
or absorbed by chemical reactions. These assays are very general,or absorbed by chemical reactions. These assays are very general,
since many reactions involve some change in heat and with use of asince many reactions involve some change in heat and with use of a
microcalorimeter, not much enzyme or substrate is required. Thesemicrocalorimeter, not much enzyme or substrate is required. These
assays can be used to measure reactions that are impossible toassays can be used to measure reactions that are impossible to
assay in any other way.assay in any other way.
 ChemiluminescentChemiluminescent ((Chemiluminescence) is the emission of lightChemiluminescence) is the emission of light
by a chemical reaction. Some enzyme reactions produce light andby a chemical reaction. Some enzyme reactions produce light and
this can be measured to detect product formation. These types ofthis can be measured to detect product formation. These types of
assay can be extremely sensitive, since the light produced can beassay can be extremely sensitive, since the light produced can be
captured by photographic film over days or weeks, but can be hardcaptured by photographic film over days or weeks, but can be hard
to quantify, because not all the light released by a reaction will beto quantify, because not all the light released by a reaction will be
detected.detected.

The detection of horseradish peroxidase by enzymaticThe detection of horseradish peroxidase by enzymatic
chemiluminescence (ECL) is a common method of detecting antibodieschemiluminescence (ECL) is a common method of detecting antibodies
in western blotting. Another example is the enzyme luciferase, this isin western blotting. Another example is the enzyme luciferase, this is
found in fireflies and naturally produces light from its substrate luciferin.found in fireflies and naturally produces light from its substrate luciferin.

AssaysAssays
 Light ScatteringLight Scattering:: measures the product ofmeasures the product of
weight-averaged molar mass and concentrationweight-averaged molar mass and concentration
of macromolecules in solution. Given a fixedof macromolecules in solution. Given a fixed
total concentration of one or more species overtotal concentration of one or more species over
the measurement time, the scattering signal is athe measurement time, the scattering signal is a
direct measure of the weight-averaged molardirect measure of the weight-averaged molar
mass of the solution, which will vary asmass of the solution, which will vary as
complexes form or dissociate. Hence thecomplexes form or dissociate. Hence the
measurement quantifies the stoichiometry of themeasurement quantifies the stoichiometry of the
complexes as well as kinetics. Light scatteringcomplexes as well as kinetics. Light scattering
assays of protein kinetics is a very generalassays of protein kinetics is a very general
technique that does not require an enzyme.technique that does not require an enzyme.

Types of Discontinuous EnzymeTypes of Discontinuous Enzyme
AssaysAssays
 RadiometricRadiometric assays measure the incorporation of radioactivity intoassays measure the incorporation of radioactivity into
substrates or its release from substrates. The radioactive isotopes mostsubstrates or its release from substrates. The radioactive isotopes most
frequently used in these assays are 14C, 32P, 35S and 125I. Sincefrequently used in these assays are 14C, 32P, 35S and 125I. Since
radioactive isotopes can allow the specific labeling of a single atom of aradioactive isotopes can allow the specific labeling of a single atom of a
substrate, these assays are both extremely sensitive and specific. They aresubstrate, these assays are both extremely sensitive and specific. They are
frequently used in biochemistry and are often the only way of measuring afrequently used in biochemistry and are often the only way of measuring a
specific reaction in crude extracts (the complex mixtures of enzymesspecific reaction in crude extracts (the complex mixtures of enzymes
produced when you lyse cells).produced when you lyse cells).
 Radioactivity is usually measured in these procedures using a scintillationRadioactivity is usually measured in these procedures using a scintillation
counter.counter.
 ChromatographicChromatographic assays measure product formation by separating theassays measure product formation by separating the
reaction mixture into its components by chromatography. This is usuallyreaction mixture into its components by chromatography. This is usually
done by high-performance liquid chromatography (HPLC), but can also usedone by high-performance liquid chromatography (HPLC), but can also use
the simpler technique of thin layer chromatography. Although this approachthe simpler technique of thin layer chromatography. Although this approach
can need a lot of material, its sensitivity can be increased by labeling thecan need a lot of material, its sensitivity can be increased by labeling the
substrates/products with a radioactive or fluorescent tag. Assay sensitivitysubstrates/products with a radioactive or fluorescent tag. Assay sensitivity
has also been increased by switching protocols to improvedhas also been increased by switching protocols to improved
chromatographic instruments (e.g. ultra-high pressure liquidchromatographic instruments (e.g. ultra-high pressure liquid
chromatography) that operate at pump pressure a few-fold higher thanchromatography) that operate at pump pressure a few-fold higher than
HPLC instrumentsHPLC instruments

Factors to control in EnzymeFactors to control in Enzyme
AssaysAssays
 Salt Concentration:Salt Concentration: Most enzymes cannot tolerate extremely high saltMost enzymes cannot tolerate extremely high salt
concentrations. The ions interfere with the weak ionic bonds of proteins.concentrations. The ions interfere with the weak ionic bonds of proteins.
Typical enzymes are active in salt concentrations of 1-500 mM. As usualTypical enzymes are active in salt concentrations of 1-500 mM. As usual
there are exceptions such as the halophilic (salt loving) algae and bacteria.there are exceptions such as the halophilic (salt loving) algae and bacteria.
 Effects of Temperature:Effects of Temperature: All enzymes work within a range of temperatureAll enzymes work within a range of temperature
specific to the organism. Increases in temperature generally lead tospecific to the organism. Increases in temperature generally lead to
increases in reaction rates. There is a limit to the increase because higherincreases in reaction rates. There is a limit to the increase because higher
temperatures lead to a sharp decrease in reaction rates. This is due to thetemperatures lead to a sharp decrease in reaction rates. This is due to the
denaturating (alteration) of protein structure resulting from the breakdown ofdenaturating (alteration) of protein structure resulting from the breakdown of
the weak ionic and hydrogen bonding that stabilize the three dimensionalthe weak ionic and hydrogen bonding that stabilize the three dimensional
structure of the enzyme. The "optimum" temperature for human enzymes isstructure of the enzyme. The "optimum" temperature for human enzymes is
usually between 35 and 40 °C. The average temperature for humans is 37usually between 35 and 40 °C. The average temperature for humans is 37
°C. Human enzymes start to denature quickly at temperatures above 40 °C.°C. Human enzymes start to denature quickly at temperatures above 40 °C.

Enzymes from thermophilic archaea found in the hot springs are stable up to 100Enzymes from thermophilic archaea found in the hot springs are stable up to 100
°C. However, the idea of an "optimum" rate of an enzyme reaction is misleading,°C. However, the idea of an "optimum" rate of an enzyme reaction is misleading,
as the rate observed at any temperature is the product of two rates, the reactionas the rate observed at any temperature is the product of two rates, the reaction
rate and the denaturation rate. If you were to use an assay measuring activity forrate and the denaturation rate. If you were to use an assay measuring activity for
one second, it would give high activity at high temperatures, however if you wereone second, it would give high activity at high temperatures, however if you were
to use an assay measuring product formation over an hour, it would give you lowto use an assay measuring product formation over an hour, it would give you low
activity at these temperatures.activity at these temperatures.

Factors to control in EnzymeFactors to control in Enzyme
AssaysAssays
 Effects of pH:Effects of pH: Most enzymes are sensitive to pH and have specificMost enzymes are sensitive to pH and have specific
ranges of activity. All have an optimum pH. The pH can stopranges of activity. All have an optimum pH. The pH can stop
enzyme activity by denaturating (altering) the three dimensionalenzyme activity by denaturating (altering) the three dimensional
shape of the enzyme by breaking ionic, and hydrogen bonds. Mostshape of the enzyme by breaking ionic, and hydrogen bonds. Most
enzymes function between a pH of 6 and 8; however pepsin in theenzymes function between a pH of 6 and 8; however pepsin in the
stomach works best at a pH of 2 and trypsin at a pH of 8.stomach works best at a pH of 2 and trypsin at a pH of 8.
 Substrate Saturation:Substrate Saturation: Increasing the substrate concentrationIncreasing the substrate concentration
increases the rate of reaction (enzyme activity). However, enzymeincreases the rate of reaction (enzyme activity). However, enzyme
saturation limits reaction rates. An enzyme is saturated when thesaturation limits reaction rates. An enzyme is saturated when the
active sites of all the molecules are occupied most of the time. Atactive sites of all the molecules are occupied most of the time. At
the saturation point, the reaction will not speed up, no matter howthe saturation point, the reaction will not speed up, no matter how
much additional substrate is added. The graph of the reaction ratemuch additional substrate is added. The graph of the reaction rate
will plateau.will plateau.
 Level of crowdingLevel of crowding, large amounts of macromolecules in a solution, large amounts of macromolecules in a solution
will alter the rates and equilibrium constants of enzyme reactions,will alter the rates and equilibrium constants of enzyme reactions,
through an effect called macromolecular crowding.through an effect called macromolecular crowding.

Immune FluorescenceImmune Fluorescence
 Most commonly, immunofluorescence employs two sets ofMost commonly, immunofluorescence employs two sets of
antibodies: a primary antibody is used against the antigen of interest;antibodies: a primary antibody is used against the antigen of interest;
a subsequent, secondary ("indirect"), dye-coupled antibody isa subsequent, secondary ("indirect"), dye-coupled antibody is
introduced that recognizes the primary antibody. In this fashion theintroduced that recognizes the primary antibody. In this fashion the
researcher may create several primary antibodies that recognizeresearcher may create several primary antibodies that recognize
various antigens, but, because they all share a common constantvarious antigens, but, because they all share a common constant
region, may be recognized by a single dye-coupled antibody.region, may be recognized by a single dye-coupled antibody.
Typically this is done by using antibodies made in different species.Typically this is done by using antibodies made in different species.
For example, a researcher might create antibodies in a goat thatFor example, a researcher might create antibodies in a goat that
recognize several antigens, and then employ dye-coupled rabbitrecognize several antigens, and then employ dye-coupled rabbit
antibodies that recognize the goat antibody constant region (denotedantibodies that recognize the goat antibody constant region (denoted
rabbit anti-goat). This allows re-use of the difficult-to-make dye-rabbit anti-goat). This allows re-use of the difficult-to-make dye-
coupled antibodies in multiple experiments.coupled antibodies in multiple experiments.
 In some cases, it is advantageous to use primary antibodies directlyIn some cases, it is advantageous to use primary antibodies directly
labelled with a fluorophore. This direct labelling decreases thelabelled with a fluorophore. This direct labelling decreases the
number of steps in the staining procedure and, more importantly,number of steps in the staining procedure and, more importantly,
often avoids cross-reactivity and high background problems.often avoids cross-reactivity and high background problems.
Fluorescent labelling can be performed in less than one hour withFluorescent labelling can be performed in less than one hour with
readily available labeling kits.readily available labeling kits.

Immune FluorescenceImmune Fluorescence
 As with most fluorescence techniques, a significant problem withAs with most fluorescence techniques, a significant problem with
immunofluorescence is photobleaching. Loss of activity caused byimmunofluorescence is photobleaching. Loss of activity caused by
photobleaching can be controlled by reducing the intensity or time-photobleaching can be controlled by reducing the intensity or time-
span of light exposure, by increasing the concentration ofspan of light exposure, by increasing the concentration of
fluorophores, or by employing more robust fluorophores that arefluorophores, or by employing more robust fluorophores that are
less prone to bleaching (e.g. Alexa Fluors or DyLight Fluors).less prone to bleaching (e.g. Alexa Fluors or DyLight Fluors).
 Many uses of immunofluorescence have been outmoded by theMany uses of immunofluorescence have been outmoded by the
development of recombinant proteins containing fluorescent proteindevelopment of recombinant proteins containing fluorescent protein
domains, e.g. green fluorescent protein (GFP). Use of such "tagged"domains, e.g. green fluorescent protein (GFP). Use of such "tagged"
proteins allows much better localization and less disruption ofproteins allows much better localization and less disruption of
protein function.protein function.

Method overviewMethod overview
1.1. In immunostaining methods, anIn immunostaining methods, an antibodyantibody is used tois used to
detect a specificdetect a specific proteinprotein epitopeepitope. These antibodies can. These antibodies can
bebe monoclonalmonoclonal oror polyclonalpolyclonal. Detection of this first or. Detection of this first or
primary antibodyprimary antibody can be accomplished in multiplecan be accomplished in multiple
ways.ways.
2.2. The primary antibody can be directly labeled using anThe primary antibody can be directly labeled using an
enzymeenzyme oror fluorophorefluorophore..
3.3. The primary antibody can be labeled using a smallThe primary antibody can be labeled using a small
molecule which interacts with a high affinity bindingmolecule which interacts with a high affinity binding
partner that can be linked to an enzyme or fluorophore.partner that can be linked to an enzyme or fluorophore.
TheThe biotinbiotin-strepavidin is one commonly used high-strepavidin is one commonly used high
affinity interaction.affinity interaction.

Method overviewMethod overview
4.4. The primary antibody can be probed for using aThe primary antibody can be probed for using a
broader species-specificbroader species-specific secondary antibodysecondary antibody that isthat is
labeled using an enzyme, or fluorophore.labeled using an enzyme, or fluorophore.
5.5. In the case ofIn the case of electron microscopyelectron microscopy, antibodies are, antibodies are
linked to a heavy metallinked to a heavy metal atomatom..
6.6. As previously described, enzymes such asAs previously described, enzymes such as horseradishhorseradish
peroxidaseperoxidase oror alkalinealkaline phosphatasephosphatase are commonlyare commonly
used to catalyse reactions that give a coloured orused to catalyse reactions that give a coloured or
chemiluminescentchemiluminescent product. Fluorescent molecules canproduct. Fluorescent molecules can
be visualised usingbe visualised using fluoresence microscopyfluoresence microscopy oror confocalconfocal
microscopymicroscopy..

ApplicationsApplications
 The applications ofThe applications of immunostainingimmunostaining areare
numerous, but are most typically used innumerous, but are most typically used in clinicalclinical
diagnosticsdiagnostics andand laboratory researchlaboratory research..
 Clinically, IHC is used inClinically, IHC is used in histopathologyhistopathology for thefor the
diagnosis of specific types of cancers based ondiagnosis of specific types of cancers based on
molecular markers.molecular markers.
 In laboratory science, immunostaining can beIn laboratory science, immunostaining can be
used for a variety of applications based onused for a variety of applications based on
investigating the presence or absence of ainvestigating the presence or absence of a
protein, its tissue distribution, its sub-cellularprotein, its tissue distribution, its sub-cellular
localization, and or changes in proteinlocalization, and or changes in protein
expression or degradationexpression or degradation

Non-Traditional (Other) MethodsNon-Traditional (Other) Methods
 Mass Spectrometry:Mass Spectrometry: A mass spectrometer creates charged particlesA mass spectrometer creates charged particles
(ions) from molecules. It then analyzes those ions to provide(ions) from molecules. It then analyzes those ions to provide
information about the molecular weight of the compound and itsinformation about the molecular weight of the compound and its
chemical structure. There are many types of mass spectrometerschemical structure. There are many types of mass spectrometers
and sample introduction techniques which allow a wide range ofand sample introduction techniques which allow a wide range of
analyses. This discussion will focus on mass spectrometry as it'sanalyses. This discussion will focus on mass spectrometry as it's
used in the powerful and widely used method of coupling Gasused in the powerful and widely used method of coupling Gas
Chromatography (GC) with Mass Spectrometry (MS).Chromatography (GC) with Mass Spectrometry (MS).
 Gas Chromatograph (GC):Gas Chromatograph (GC): A mixture of compounds to be analysedA mixture of compounds to be analysed
is initially injected into the GC where the mixture is vaporized in ais initially injected into the GC where the mixture is vaporized in a
heated chamber. The gas mixture travels through a GC column,heated chamber. The gas mixture travels through a GC column,
where the compounds become separated as they interact with thewhere the compounds become separated as they interact with the
column. The chromatogram on the right shows peaks which resultcolumn. The chromatogram on the right shows peaks which result
from this separation. Those separated compounds then immediatelyfrom this separation. Those separated compounds then immediately
enter the mass spectrometer.enter the mass spectrometer.

Mass Spectrometer (MS)Mass Spectrometer (MS)
 All massAll mass
spectrometers consistspectrometers consist
of three distinctof three distinct
regions.regions.
1) Ionizer1) Ionizer
2) Ion analyzer2) Ion analyzer
3) Detector3) Detector

IonizerIonizer
 In the GC-MS discussed in this introduction, the charged particles (ions)In the GC-MS discussed in this introduction, the charged particles (ions)
required for mass analysis are formed by Electron Impact (EI) Ionization.required for mass analysis are formed by Electron Impact (EI) Ionization.
The gas molecules exiting the GC are bombarded by a high-energy electronThe gas molecules exiting the GC are bombarded by a high-energy electron
beam (70 eV). An electron which strikes a molecule may impart enoughbeam (70 eV). An electron which strikes a molecule may impart enough
energy to remove another electron from that molecule. Methanol, forenergy to remove another electron from that molecule. Methanol, for
example, would undergo the following reaction in the ionizing region:example, would undergo the following reaction in the ionizing region:
CH3OH + 1 electron CH3OH+CH3OH + 1 electron CH3OH+..+ 2 electrons+ 2 electrons
(note: the symbols(note: the symbols +.+. indicate that a radical cation was formed)indicate that a radical cation was formed)
 EI Ionization usually produces singly charged ions containing one unpairedEI Ionization usually produces singly charged ions containing one unpaired
electron. A charged molecule which remains intact is called the molecularelectron. A charged molecule which remains intact is called the molecular
ion. Energy imparted by the electron impact and, more importantly,ion. Energy imparted by the electron impact and, more importantly,
instability in a molecular ion can cause that ion to break into smaller piecesinstability in a molecular ion can cause that ion to break into smaller pieces
(fragments). The methanol ion may fragment in various ways, with one(fragments). The methanol ion may fragment in various ways, with one
fragment carrying the charge and one fragment remaining uncharged. Forfragment carrying the charge and one fragment remaining uncharged. For
example:example:
CH3OH+CH3OH+..(molecular ion) CH2OH+(fragment ion) + H(molecular ion) CH2OH+(fragment ion) + H..
(or) CH3OH+(or) CH3OH+..(molecular ion) CH3+(fragment ion) +(molecular ion) CH3+(fragment ion) + ..OHOH

Ion AnalyzerIon Analyzer
 Molecular ions and fragment ions are accelerated by manipulationMolecular ions and fragment ions are accelerated by manipulation
of the charged particles through the mass spectrometer.of the charged particles through the mass spectrometer.
Uncharged molecules and fragments are pumped away. TheUncharged molecules and fragments are pumped away. The
quadrupole mass analyzer in this example uses positive (+) andquadrupole mass analyzer in this example uses positive (+) and
negative (-) voltages to control the path of the ions. Ions travelnegative (-) voltages to control the path of the ions. Ions travel
down the path based on their mass to charge ratio (m/z). EIdown the path based on their mass to charge ratio (m/z). EI
ionization produces singly charged particles, so the charge (z) isionization produces singly charged particles, so the charge (z) is
one. Therefore an ion's path will depend on its mass. If the (+) andone. Therefore an ion's path will depend on its mass. If the (+) and
(-) rods shown in the mass spectrometer schematic were ?fixed' at a(-) rods shown in the mass spectrometer schematic were ?fixed' at a
particular rf/dc voltage ratio, then one particular m/z would travel theparticular rf/dc voltage ratio, then one particular m/z would travel the
successful path shown by the solid line to the detector. However,successful path shown by the solid line to the detector. However,
voltages are not fixed, but are scanned so that ever increasingvoltages are not fixed, but are scanned so that ever increasing
masses can find a successful path through the rods to the detector.masses can find a successful path through the rods to the detector.

DetectorDetector
 There are many types of detectors, but most work byThere are many types of detectors, but most work by
producing an electronic signal when struck by an ion.producing an electronic signal when struck by an ion.
Timing mechanisms which integrate those signals withTiming mechanisms which integrate those signals with
the scanning voltages allow the instrument to reportthe scanning voltages allow the instrument to report
which m/z strikes the detector. The mass analyzer sortswhich m/z strikes the detector. The mass analyzer sorts
the ions according to m/z and the detector records thethe ions according to m/z and the detector records the
abundance of each m/z. Regular calibration of the m/zabundance of each m/z. Regular calibration of the m/z
scale is necessary to maintain accuracy in thescale is necessary to maintain accuracy in the
instrument. Calibration is performed by introducing ainstrument. Calibration is performed by introducing a
well known compound into the instrument and "tweaking"well known compound into the instrument and "tweaking"
the circuits so that the compound's molecular ion andthe circuits so that the compound's molecular ion and
fragment ions are reported accurately.fragment ions are reported accurately.

Protein Structure PredictionProtein Structure Prediction
 is the prediction of the three-dimensionalis the prediction of the three-dimensional
structure of a protein from its amino acidstructure of a protein from its amino acid
sequence—that is, the prediction of a protein'ssequence—that is, the prediction of a protein's
tertiary structure from its primary structure. It istertiary structure from its primary structure. It is
one of the most important goals pursued byone of the most important goals pursued by
bioinformatics and theoretical chemistry. Proteinbioinformatics and theoretical chemistry. Protein
structure prediction is of high importance instructure prediction is of high importance in
medicine (for example, in drug design) andmedicine (for example, in drug design) and
biotechnology (for example, in the design ofbiotechnology (for example, in the design of
novel enzymes).novel enzymes).

Protein SequencingProtein Sequencing
 Proteins are found in every cell and are essential to every biologicalProteins are found in every cell and are essential to every biological
process, protein structure is very complex: determining a protein'sprocess, protein structure is very complex: determining a protein's
structure involves firststructure involves first protein sequencingprotein sequencing - determining the amino- determining the amino
acid sequences of its constituent peptides; and also determiningacid sequences of its constituent peptides; and also determining
what conformation it adopts and whether it is complexed with anywhat conformation it adopts and whether it is complexed with any
non-peptide molecules. Discovering the structures and functions ofnon-peptide molecules. Discovering the structures and functions of
proteins in living organisms is an important tool for understandingproteins in living organisms is an important tool for understanding
cellular processes, and allows drugs that target specific metaboliccellular processes, and allows drugs that target specific metabolic
pathways to be invented more easily.pathways to be invented more easily.
 The two major direct methods of protein sequencing areThe two major direct methods of protein sequencing are massmass
spectrometryspectrometry and theand the Edman degradation reactionEdman degradation reaction. It is also. It is also
possible to generate an amino acid sequence from the DNA orpossible to generate an amino acid sequence from the DNA or
mRNA sequence encoding the protein, if this is known. However,mRNA sequence encoding the protein, if this is known. However,
there are a number of other reactions which can be used to gainthere are a number of other reactions which can be used to gain
more limited information about protein sequences and can be usedmore limited information about protein sequences and can be used
as preliminaries to the aforementioned methods of sequencing or toas preliminaries to the aforementioned methods of sequencing or to
overcome specific inadequacies within them.overcome specific inadequacies within them.

 Edman degradationEdman degradation: is a method of: is a method of
sequencing amino acids in a peptide. Insequencing amino acids in a peptide. In
this method, the amino-terminal residue isthis method, the amino-terminal residue is
labeled and cleaved from the peptidelabeled and cleaved from the peptide
without disrupting the peptide bondswithout disrupting the peptide bonds
between other amino acid residues.between other amino acid residues.

 PhenylisothiocyanatePhenylisothiocyanate is reacted with an uncharged terminal aminois reacted with an uncharged terminal amino
group, under mildly alkaline conditions, to form a cyclicalgroup, under mildly alkaline conditions, to form a cyclical
phenylthiocarbamoylphenylthiocarbamoyl derivative. Then, under acidic conditions, thisderivative. Then, under acidic conditions, this
derivative of the terminal amino acid is cleaved as a thiazolinonederivative of the terminal amino acid is cleaved as a thiazolinone
derivative. The thiazolinone amino acid is then selectively extractedderivative. The thiazolinone amino acid is then selectively extracted
into an organic solvent and treated with acid to form the more stableinto an organic solvent and treated with acid to form the more stable
phenylthiohydantoin (PTH)- amino acid derivative that can bephenylthiohydantoin (PTH)- amino acid derivative that can be
identified by using chromatography or electrophoresis.identified by using chromatography or electrophoresis.
 This procedure can then be repeated again to identify the next aminoThis procedure can then be repeated again to identify the next amino
acid. A major drawback to this technique is that the peptides beingacid. A major drawback to this technique is that the peptides being
sequenced in this manner cannot have more than 50 to 60 residuessequenced in this manner cannot have more than 50 to 60 residues
(and in practice, under 30). The peptide length is limited due to the(and in practice, under 30). The peptide length is limited due to the
cyclical derivitization not always going to completion. Thecyclical derivitization not always going to completion. The
derivitization problem can be resolved by cleaving large peptides intoderivitization problem can be resolved by cleaving large peptides into
smaller peptides before proceeding with the reaction. It is able tosmaller peptides before proceeding with the reaction. It is able to
accurately sequence up to 30 amino acids with modern machinesaccurately sequence up to 30 amino acids with modern machines
capable of over 99% efficiency per amino acid. An advantage of thecapable of over 99% efficiency per amino acid. An advantage of the
Edman degradation is that it only uses 10 - 100 picomoles of peptideEdman degradation is that it only uses 10 - 100 picomoles of peptide
for the sequencing process. Edman degradation reaction isfor the sequencing process. Edman degradation reaction is
automated to speed up the process.automated to speed up the process.

Protein Structural AlignmentsProtein Structural Alignments
 Structural alignmentStructural alignment is a form of sequence alignment that is basedis a form of sequence alignment that is based
on comparison of shape. These alignments attempt to establishon comparison of shape. These alignments attempt to establish
equivalences between two or more polymer structures based onequivalences between two or more polymer structures based on
their shape and three-dimensional conformation. This process istheir shape and three-dimensional conformation. This process is
usually applied to protein tertiary structures but can also be used forusually applied to protein tertiary structures but can also be used for
large RNA molecules. In contrast to simple structural superposition,large RNA molecules. In contrast to simple structural superposition,
where at least some equivalent residues of the two structures arewhere at least some equivalent residues of the two structures are
known, structural alignment requires noknown, structural alignment requires no a prioria priori knowledge ofknowledge of
equivalent positions. Structural alignment is a valuable tool for theequivalent positions. Structural alignment is a valuable tool for the
comparison of proteins with low sequence similarity, wherecomparison of proteins with low sequence similarity, where
evolutionary relationships between proteins cannot be easilyevolutionary relationships between proteins cannot be easily
detected by standard sequence alignment techniques. Structuraldetected by standard sequence alignment techniques. Structural
alignment can therefore be used to imply evolutionary relationshipsalignment can therefore be used to imply evolutionary relationships
between proteins that share very little common sequence. However,between proteins that share very little common sequence. However,
caution should be used in using the results as evidence for sharedcaution should be used in using the results as evidence for shared
evolutionary ancestry because of the possible confounding effectsevolutionary ancestry because of the possible confounding effects
of convergent evolution by which multiple unrelated amino acidof convergent evolution by which multiple unrelated amino acid
sequences converge on a common tertiary structure.sequences converge on a common tertiary structure.

 Structural alignments can compare two sequences orStructural alignments can compare two sequences or multiple sequencesmultiple sequences..
Because these alignments rely on information about all the queryBecause these alignments rely on information about all the query
sequences' three-dimensional conformations, the method can only be usedsequences' three-dimensional conformations, the method can only be used
on sequences where these structures are known. These are usually foundon sequences where these structures are known. These are usually found
byby X-ray crystallographyX-ray crystallography oror NMR spectroscopyNMR spectroscopy. It is possible to perform a. It is possible to perform a
structural alignment on structures produced bystructural alignment on structures produced by structure predictionstructure prediction methods.methods.
Indeed, evaluating such predictions often requires a structural alignmentIndeed, evaluating such predictions often requires a structural alignment
between the model and the true known structure to assess the model'sbetween the model and the true known structure to assess the model's
quality. Structural alignments are especially useful in analyzing data fromquality. Structural alignments are especially useful in analyzing data from
structuralstructural genomicsgenomics andand proteomicsproteomics efforts, and they can be used asefforts, and they can be used as
comparison points to evaluate alignments produced by purely sequence-comparison points to evaluate alignments produced by purely sequence-
basedbased bioinformaticsbioinformatics methods.methods.
 The outputs of a structural alignment are a superposition of the atomicThe outputs of a structural alignment are a superposition of the atomic
coordinate setscoordinate sets and a minimaland a minimal root mean squareroot mean square distance (distance (RMSDRMSD) between) between
the structures. The RMSD of two aligned structures indicates theirthe structures. The RMSD of two aligned structures indicates their
divergence from one another. Structural alignment can be complicated bydivergence from one another. Structural alignment can be complicated by
the existence of multiplethe existence of multiple protein domainsprotein domains within one or more of the inputwithin one or more of the input
structures, because changes in relative orientation of the domains betweenstructures, because changes in relative orientation of the domains between
two structures to be aligned can artificially inflate the RMSD.two structures to be aligned can artificially inflate the RMSD.

METHODS:METHODS:
 GANGSTAGANGSTA ((GGeneticenetic AAlgorithm forlgorithm for NNon-sequential,on-sequential,
GGapped proteinapped protein STSTructureructure AAlignment) is a method forlignment) is a method for
non-sequential protein structure alignment using a two-non-sequential protein structure alignment using a two-
level hierarchical approach. On the first level, pairwiselevel hierarchical approach. On the first level, pairwise
contacts and relative orientations between SSEs arecontacts and relative orientations between SSEs are
maximized using a genetic algorithm (GA) and proteinmaximized using a genetic algorithm (GA) and protein
graph representation. On the second level, pairwisegraph representation. On the second level, pairwise
residue contact maps resulting from the best SSEresidue contact maps resulting from the best SSE
alignments are optimized. GANGSTA can be used onlinealignments are optimized. GANGSTA can be used online
atat http://agknapp.chemie.fu-berlin.de/gangstahttp://agknapp.chemie.fu-berlin.de/gangsta

METHODS:METHODS:
 MAMMOTH (MAMMOTH (MAtching Molecular Models ObtainedMAtching Molecular Models Obtained
from Theory)from Theory) -based structure alignment methods-based structure alignment methods
decomposes the protein structure into short peptidesdecomposes the protein structure into short peptides
(heptapeptides) which are compared with the(heptapeptides) which are compared with the
heptapeptides of another protein. Similarity scoreheptapeptides of another protein. Similarity score
between two heptapeptides is calculated using a unit-between two heptapeptides is calculated using a unit-
vector RMS (URMS) method. These scores are stored invector RMS (URMS) method. These scores are stored in
a similarity matrix, and with a hybrid (local-global)a similarity matrix, and with a hybrid (local-global)
dynamic programming the optimal residue alignment isdynamic programming the optimal residue alignment is
calculated. Protein similarity scores calculated withcalculated. Protein similarity scores calculated with
MAMMOTH is derived from the likelihood of obtaining aMAMMOTH is derived from the likelihood of obtaining a
given structural alignment by chancegiven structural alignment by chance

 RAPIDORAPIDO (R(Rapidapid AAlignment oflignment of PProteinsroteins IIn terms ofn terms of DoDomains) is a web servermains) is a web server
for the 3D alignment of crystal structures of different protein molecules, in thefor the 3D alignment of crystal structures of different protein molecules, in the
presence of conformational changes. RAPIDO identifies fragments that arepresence of conformational changes. RAPIDO identifies fragments that are
structurally similar in the two proteins using an approach based on differencestructurally similar in the two proteins using an approach based on difference
distance matrices. The Matching Fragment Pairs (MFPs) are thendistance matrices. The Matching Fragment Pairs (MFPs) are then
represented as nodes in a graph which are chained together to form anrepresented as nodes in a graph which are chained together to form an
alignment by means of an algorithm for the identification of the longest pathalignment by means of an algorithm for the identification of the longest path
on a DAG (Directed Acyclic Graph). The final step of refinement is performedon a DAG (Directed Acyclic Graph). The final step of refinement is performed
to improve the quality of the alignment. After aligning the two structures theto improve the quality of the alignment. After aligning the two structures the
server applies a genetic algorithm for the identification of conformationallyserver applies a genetic algorithm for the identification of conformationally
invariant regions . These regions correspond to groups of atoms whoseinvariant regions . These regions correspond to groups of atoms whose
interatomic distances are constant (within a defined tolerance). In doing sointeratomic distances are constant (within a defined tolerance). In doing so
RAPIDO takes into account the variation in the reliability of atomicRAPIDO takes into account the variation in the reliability of atomic
coordinates by employing weighting-functions based on the refined B-values.coordinates by employing weighting-functions based on the refined B-values.
The regions identified as conformationally invariant by RAPIDO representThe regions identified as conformationally invariant by RAPIDO represent
reliable sets of atoms for the superposition of the two structures that can bereliable sets of atoms for the superposition of the two structures that can be
used for a detailed analysis of changes in the conformation. In addition to theused for a detailed analysis of changes in the conformation. In addition to the
functionalities provided by existing tools, RAPIDO can identify structurallyfunctionalities provided by existing tools, RAPIDO can identify structurally
equivalent regions even when these consist of fragments that are distant inequivalent regions even when these consist of fragments that are distant in
terms of sequence and separated by other movable domains.terms of sequence and separated by other movable domains.

 SABERTOOTHSABERTOOTH uses structural profiles to perform structuraluses structural profiles to perform structural
alignments. The underlying structural profiles expresses the globalalignments. The underlying structural profiles expresses the global
connectivity of each residue. Despite the very condensed vectorialconnectivity of each residue. Despite the very condensed vectorial
representation, the tool recognizes structural similarities withrepresentation, the tool recognizes structural similarities with
accuracy comparable to established alignment tools based onaccuracy comparable to established alignment tools based on
coordinates and performs comparably in quality. Furthermore, thecoordinates and performs comparably in quality. Furthermore, the
algorithm has favourable scaling of computation time with chainalgorithm has favourable scaling of computation time with chain
length. Since the algorithm is independent of the details of thelength. Since the algorithm is independent of the details of the
structural representation, the framework can be generalized tostructural representation, the framework can be generalized to
sequence-to-sequence and sequence-to-structure comparisonsequence-to-sequence and sequence-to-structure comparison
within the same setup, and it is therefore more general than otherwithin the same setup, and it is therefore more general than other
tools. SABERTOOTH can be used online at http://www.fkp.tu-tools. SABERTOOTH can be used online at http://www.fkp.tu-
darmstadt.de/sabertooth/darmstadt.de/sabertooth/

Peptide Mass FingerprintingPeptide Mass Fingerprinting
 Peptide mass fingerprintingPeptide mass fingerprinting (PMF) (also known as(PMF) (also known as
protein fingerprintingprotein fingerprinting) is an analytical technique for) is an analytical technique for
protein identification that was developed in 1993 byprotein identification that was developed in 1993 by
several groups independently. In this method, theseveral groups independently. In this method, the
unknown protein of interest is first cleaved into smallerunknown protein of interest is first cleaved into smaller
peptidespeptides, whose absolute masses can be accurately, whose absolute masses can be accurately
measured with ameasured with a mass spectrometermass spectrometer such assuch as
MALDI-TOFMALDI-TOF oror ESI-TOFESI-TOF. These masses are then. These masses are then inin
silicosilico compared to either a database containing knowncompared to either a database containing known
protein sequences or even the genome. This is achievedprotein sequences or even the genome. This is achieved
by using computer programs that translate the knownby using computer programs that translate the known
genome of the organism into proteins, then theoreticallygenome of the organism into proteins, then theoretically
cut the proteins into peptides, and calculate the absolutecut the proteins into peptides, and calculate the absolute
masses of the peptides from each protein. They thenmasses of the peptides from each protein. They then
compare the masses of the peptides of the unknowncompare the masses of the peptides of the unknown
protein to the theoretical peptide masses of each proteinprotein to the theoretical peptide masses of each protein
encoded in the genome. The results are statisticallyencoded in the genome. The results are statistically
analyzed to find the best match.analyzed to find the best match.

Peptide Mass FingerprintingPeptide Mass Fingerprinting
 The advantage of this method is that only the masses of theThe advantage of this method is that only the masses of the
peptides have to be known and as such,peptides have to be known and as such, de novode novo
peptide sequencingpeptide sequencing is not necessary which can be time consuming.is not necessary which can be time consuming.
A disadvantage is that the protein sequence has to be present in theA disadvantage is that the protein sequence has to be present in the
database of interest. Additionally most PMF algorithms assume thatdatabase of interest. Additionally most PMF algorithms assume that
the peptides come from a single protein. The presence of a mixturethe peptides come from a single protein. The presence of a mixture
can significantly complicate the analysis and potentially compromisecan significantly complicate the analysis and potentially compromise
the results. Typical for the PMF based protein identification is thethe results. Typical for the PMF based protein identification is the
requirement for an isolated protein. Mixtures exceeding a number ofrequirement for an isolated protein. Mixtures exceeding a number of
2-3 proteins typically require the additional use of2-3 proteins typically require the additional use of MS/MSMS/MS basedbased
protein identification to achieve sufficient specificity of identification.protein identification to achieve sufficient specificity of identification.
Therefore, the typical PMF samples are isolated proteins fromTherefore, the typical PMF samples are isolated proteins from
Two-dimensional gel electrophoresisTwo-dimensional gel electrophoresis (2D gels) or isolated(2D gels) or isolated
SDS-PAGESDS-PAGE bands. Additional analyses bybands. Additional analyses by MS/MSMS/MS can either becan either be
direct, e.g., MALDI-TOF/TOF analysis or downstream nanoLC-ESI-direct, e.g., MALDI-TOF/TOF analysis or downstream nanoLC-ESI-
MS/MS analysis of gel spot eluates.MS/MS analysis of gel spot eluates.

Ligand Binding AssayLigand Binding Assay
 aa ligand binding assayligand binding assay is technique to identifyis technique to identify
the presence of a molecule and quantify it.the presence of a molecule and quantify it.
 Historically, it was used to test for estrogenHistorically, it was used to test for estrogen
receptor and progesterone receptor in breastreceptor and progesterone receptor in breast
tumorstumors
 Characterization through ligand interaction mayCharacterization through ligand interaction may
be performed by electroblotting or by affinitybe performed by electroblotting or by affinity
electrophoresis in agarose or by capillaryelectrophoresis in agarose or by capillary
electrophoresis as for estimation of bindingelectrophoresis as for estimation of binding
constants and determination of structuralconstants and determination of structural
features like glycan content through lectinfeatures like glycan content through lectin
binding.binding.

REFERENCESREFERENCES
1.1. Adessi, C., Miege, C., Albrieux, C. & Rabilloud, T. (1997) Two-dimensionalAdessi, C., Miege, C., Albrieux, C. & Rabilloud, T. (1997) Two-dimensional
electrophoresis of membrane proteins: A current challenge for immobilized pHelectrophoresis of membrane proteins: A current challenge for immobilized pH
gradients. Electrophoresis, 18, 127-135gradients. Electrophoresis, 18, 127-135
2.2. Bergmeyer, H. U. "Methods of Enzymatic Analysis", Vol. 4, Academic Press (NewBergmeyer, H. U. "Methods of Enzymatic Analysis", Vol. 4, Academic Press (New
York, NY:1974), pp.2066-2072.York, NY:1974), pp.2066-2072.
3.3. Coons, Albert (1941). "Immunological properties of an antibody containing aCoons, Albert (1941). "Immunological properties of an antibody containing a
fluorescent group". Proc Soc Exp Biol Med 47: 200–202.fluorescent group". Proc Soc Exp Biol Med 47: 200–202.
4.4. Cherie, H (2004). "Applications of Flow Cytometry and Immunohistochemistry toCherie, H (2004). "Applications of Flow Cytometry and Immunohistochemistry to
Diagnostic Hematopathology". Archives of Pathology and Laboratory Medicine 128Diagnostic Hematopathology". Archives of Pathology and Laboratory Medicine 128
(9): 1004–1022.(9): 1004–1022.
5.5. Churchwella, M; Twaddlea, N; Meekerb, L; and Doergea, D. Improving Sensitivity InChurchwella, M; Twaddlea, N; Meekerb, L; and Doergea, D. Improving Sensitivity In
Liquid Chromatography-Mass Spectrometry. Journal of Chromatography B VolumeLiquid Chromatography-Mass Spectrometry. Journal of Chromatography B Volume
825, Issue 2, 25 October 2005, Pages 134-143 Layne, E. Spectrophotometric and825, Issue 2, 25 October 2005, Pages 134-143 Layne, E. Spectrophotometric and
Turbidimetric Methods for Measuring Proteins.Turbidimetric Methods for Measuring Proteins. Methods in Enzymology 3:Methods in Enzymology 3: 447-455.447-455.
1957.1957.
6.6. Edman, P. Acta Chem. Scand. 1950, 4, 283.Edman, P. Acta Chem. Scand. 1950, 4, 283.
7.7. Görg, A., Postel, W. & Gunther, S. (1988) The current state of two-dimensionalGörg, A., Postel, W. & Gunther, S. (1988) The current state of two-dimensional
electrophoresis with immobilized pH gradients. Electrophoresis, 9, 531-546.electrophoresis with immobilized pH gradients. Electrophoresis, 9, 531-546.
8.8. Görg, A., Obermaier, C., Boguth, G., Harder, A., Scheibe, B., Wildgruber, R. &Görg, A., Obermaier, C., Boguth, G., Harder, A., Scheibe, B., Wildgruber, R. &
Weiss, W. (2000) The current state of two-dimensional electrophoresis withWeiss, W. (2000) The current state of two-dimensional electrophoresis with
immobilized pH gradients. Electrophoresis, 21, 1037-1053.immobilized pH gradients. Electrophoresis, 21, 1037-1053.
9.9. Lodish H, Berk A, Matsudaira P, et al. (2004). Molecular Cell Biology (5th ed.). WHLodish H, Berk A, Matsudaira P, et al. (2004). Molecular Cell Biology (5th ed.). WH
Freeman: New York, NYFreeman: New York, NY

REFERENCESREFERENCES
10.10. Niall HD (1973). "Automated Edman degradation: the protein sequenator". Meth. Enzymol. 27: 942–1010.Niall HD (1973). "Automated Edman degradation: the protein sequenator". Meth. Enzymol. 27: 942–1010.
11.11. Kedem, K., Chew, L., and Elber, R. 1999. Unit-vector RMS (URMS) as a tool to analyze molecular dynamicsKedem, K., Chew, L., and Elber, R. 1999. Unit-vector RMS (URMS) as a tool to analyze molecular dynamics
trajectories. Proteins 37: 554–564trajectories. Proteins 37: 554–564
12.12. Mosca, Schneider TR. (2008) RAPIDO: a web server for the alignment of protein structures in the presence ofMosca, Schneider TR. (2008) RAPIDO: a web server for the alignment of protein structures in the presence of
conformational changes, Nucleic Acids Research, doi: 10.1093/nar/gkn197 Open Access RAPIDO serverconformational changes, Nucleic Acids Research, doi: 10.1093/nar/gkn197 Open Access RAPIDO server
13.13. Pappin DJ, Hojrup P, Bleasby AJ (1993). "Rapid identification of proteins by peptide-mass fingerprinting".Pappin DJ, Hojrup P, Bleasby AJ (1993). "Rapid identification of proteins by peptide-mass fingerprinting".
Curr. Biol. 3 (6): 327–32Curr. Biol. 3 (6): 327–32
14.14. Ramos-Vara, JA (2005). "Technical Aspects of Immunohistochemistry". Vet Pathol 42: 405–426Ramos-Vara, JA (2005). "Technical Aspects of Immunohistochemistry". Vet Pathol 42: 405–426
15.15. Righetti, P.G (1983) Isoelectric focusing: theory, methodology and applications. Laboratory techniques inRighetti, P.G (1983) Isoelectric focusing: theory, methodology and applications. Laboratory techniques in
biochemistry and molecular biology, vol. 11. Eds Work, T.S. & Burdon, R.H. Elsevier Biomedical Press,biochemistry and molecular biology, vol. 11. Eds Work, T.S. & Burdon, R.H. Elsevier Biomedical Press,
Amsterdam, Netherlands.Amsterdam, Netherlands.
16.16. Righetti, P.G (1990) Immobilized pH gradients: theory and methodology. Laboratory techniques inRighetti, P.G (1990) Immobilized pH gradients: theory and methodology. Laboratory techniques in
biochemistry and molecular biology, vol. 20. Eds Burdon, R.H. & van Knippenberg, P.H., Elsevier Biomedicalbiochemistry and molecular biology, vol. 20. Eds Burdon, R.H. & van Knippenberg, P.H., Elsevier Biomedical
Press, Amsterdam, Netherlands.Press, Amsterdam, Netherlands.
17.17. Schnell, S., Chappell, M. J., Evans, N. D. and Roussel, M. R. The mechanism distinguishability problem inSchnell, S., Chappell, M. J., Evans, N. D. and Roussel, M. R. The mechanism distinguishability problem in
biochemical kinetics: The single-enzyme, single-substrate reaction as a case study.biochemical kinetics: The single-enzyme, single-substrate reaction as a case study. Comptes RendusComptes Rendus
BiologiesBiologies 2006; 329, 51-612006; 329, 51-61
18.18. Stoscheck, CM. Quantitation of Protein.Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182:Methods in Enzymology 182: 50-69. 1990.50-69. 1990.
19.19. Todd MJ, Gomez J. Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? AnalTodd MJ, Gomez J. Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? Anal
Biochem. 2001 Sep 15;296(2):179-87.Biochem. 2001 Sep 15;296(2):179-87.
20.20. www.shsu.edu/~chm_tgc/sounds/sound.htmlwww.shsu.edu/~chm_tgc/sounds/sound.html
21.21. Zhang Y, Skolnick J. (2005). The protein structure prediction problem could be solved using the current PDB library. Proc Natl AcadZhang Y, Skolnick J. (2005). The protein structure prediction problem could be solved using the current PDB library. Proc Natl Acad
Sci USA 102(4):1029-34.Sci USA 102(4):1029-34.

Protein Detection Methods and Application

More Related Content

What's hot (20)

Similar to Protein Detection Methods and Application (20)

More from angelsalaman (10)

Recently uploaded (20)

Protein Detection Methods and Application