Basics of protein biochemistry
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Buffers resist changes in pH and consist of a conjugate acid-base pair where the ratio of proton acceptor to proton donor is near unity. A webinar discusses how biological buffers can impact reproducibility and explores considerations for buffer selection and use to improve reproducibility. Key factors include how buffers may interact with metals and biological materials and tips for proper buffer selection and use. [END SUMMARY]
Basics of protein biochemistry
- 1. BASICS OF PROTEIN BIOCHEMISTRY 26th Sep ’ 2018
- 2. BUFFER An aqueous solution capable of resisting changes in pH, consisting of a conjugate acid- base pair in which the ratio of proton acceptor to proton donor is near unity. Eg: bicarbonate buffer, tris buffer
- 4. Webinar: Improving Reproducibility with Biological Buffers Abstract Recent publications highlight the low reproducibility rate of preclinical research and the issues that surround it.Various sources for this irreproducibility can be grouped into four main categories: study design, laboratory protocols, data analysis and reporting, and the actual biological reagents and reference materials used. Improvement efforts for biological reagents have primarily focused on validating antibodies and authenticating cell lines, two areas where significant improvement opportunities exist. What about other agents like buffers?These ubiquitous life science products are often overlooked as a source of variability in experiments, yet they can have a significant contribution to reproducibility.Since buffers are so widely used across most life science applications and workflows, there is a need for better understanding and training on their selection and use. Watch this informative webinar in which our experts will explore ways to improve reproducibility when using biological buffers. What WillYou Learn? Important considerations when using biological buffers, like how strongly buffers may interact with metals and other biological materials Tools available to help make buffer selection easier – how to select the right quality and grade for a given application Other practical tips to improve reproducibility with the use of biological buffers Who ShouldWatch? Lab Managers Principle Investigators Process Scientists/ Manufacturing /Production R&D Scientists Clinical Researchers Regulatory/QA/QC/Validation Professionals Graduate Students Procurement Professionals techniquesimproving-reproducibility-with-biological-buffers-webinar.mp4 https://www.sigmaaldrich.com/video/life- science/improving-reproducibility-with- biological-buffers.html
- 5. What is Molarity? Mass (g) = Concentration (mol/L) x Volume (L) x Molecular Weight (g/mol)
- 6. What is Dilution? Dilution is the process of reducing the concentration of a solute in solution, usually simply by mixing with more solvent C1V1 = C2V2 where: V1 = Volume of stock solution needed to make the new solution C1 = Concentration of stock solution V2 = Final volume of new solution C2 = Final concentration of new solution Example: Make 5 mL of a 0.25 M solution from a 1 M solution (V1)(1 M) = (5 mL)(0.25 M) V1 = 1.25 mL Answer: Place 1.25 mL of the 1 M solution into V1-V2 = 5 mL – 1.25 mL = 3.75 mL of diluent
- 7. SDS-PAGE SDS-PAGE (sodium dodecyl sulphate- polyacrylamide gel electrophoresis) is a technique commonly used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids based on their molecular weight. SDS is an anionic detergent, where the negative charges destroy most of the complex structure of proteins and are strongly attracted toward an anode (positively-charged electrode) in an electric field. Polyacrylamide gels restrain larger molecules from migrating as fast as smaller molecules.
- 8. Steps in SDS-PAGE Extract Protein Solubilize and Denature Protein by detergents and sonication Separate Proteins on a gel Stain proteins (visualization) Analyze and interpret results
- 9. Things to do before SDS-PAGE Cells Containing Protein Cell Lysis by Detergents and Sonication Heat Denaturation of Proteins + - Load Proteins on Gel Apply Electric Current - - - - - Proteins Separate by Size Detergents Bind with Proteins - - - - - - - - - - Bacterial culture Harvesting IMAC = elution of proteins
- 10. The separation of proteins involves the use of polyacrylamide as the matrix. Polymerization of acrylamide monomers is induced by ammonium persulfate (APS), which spontaneously decomposes to form free radicals. Tetramethylethylenediamine (TEMED), a catalyst generally used to promote polymerization. Boiling the protein in the presence of the detergent sodium dodecyl sulfate (SDS) and the reducing agent b-mercaptoethanol (which reduces disulfide bonds) results in disruption of the complex structure of the protein.
- 11. The free radicals transfer electrons to the acrylamide monomers, radicalizing them and causing them to react with each other to form the polyacrylamide chain. In the absence of bis-acrylamide, the acrylamide would polymerize into long strands (linear polymer ), not a porous gel.
- 12. SDS (the detergent soap) breaks up hydrophobic areas and coats proteins with negative charges thus overwhelms any intrinsic charge present in the protein. The detergent binds to hydrophobic regions approximately one molecule of SDS for every two amino acid residues.
- 13. o The treatment with SDS and b-mercaptoethanol will result in the formation of denatured protein monomers; it is these protein monomers that are separated on the SDS PAGE. o The molecular weight standards can be used to calibrate the migration of proteins of differing sizes on the gel. o The proteins within an SDS polyacrylamide gel are denatured; the molecular weight determined will be that of the individual monomers of multimeric proteins.
- 15. o Protein denature is usually done by boiling the protein sample in SDS PAGE sample buffer. o The sample buffer has three roles: The sample buffer provides the SDS necessary for the uniform charge-to-mass ratio. The sample buffer has bromophenol blue, a blue dye that runs at a low apparent molecular weight.
- 16. Finally, the sample buffer contains glycerol to make the protein sample denser than the electrophoresis tank buffer, so that the protein sample will sink to the bottom of the well when you load it. The type of SDS polyacrylamide gel you will use consists of two layers: The top layer is the stacking gel and the bottom layer is the resolving gel (separating gel ) , based on a method invented by Laemmli in 1970. The stacking gel concentrates all of the protein in a narrow region, while the resolving gel performs the actual separation of the proteins by molecular weight.
- 17. o The stacking gel is prepared at a lower pH (6.8) and lower acrylamide percentage . • In the absence of a stacking gel, your sample would sit on top of the running gel, as a band of up to 1cm deep rather than being lined up together and hitting the running gel together, the proteins in the sample would all enter the running gel at different times, resulting in very smeared bands . • So the stacking gel, ensures that all of the proteins arrive at the running gel at the same time so proteins of the same molecular weight will migrate as tight bands.
- 18. The resolving gel ( Separation) is at pH 8.8 and has the desired acrylamide concentration for separation of proteins in the appropriate size range . Once the proteins are in the running gel, they are separated because higher molecular weight proteins move more slowly through the porous acrylamide gel than lower molecular weight proteins.
- 19. • When the power is turned on: 1. Negatively-charged glycine ions in the pH 8.3 electrode buffer are forced to enter the stacking gel, where the pH is 6.8. In this environment glycine switches predominantly to the zwitterionic (neutrally charged) state. This loss of charge causes them to move very slowly in the electric field. 2. The Cl- ions (from Tris-HCl) on the other hand, move much more quickly in the electric field and they form an ion front that migrates ahead of the glycine.
- 20. o 3. The SDS-coated protein molecules and the dye, which have charge-to-mass ratios greater than that of the glycine but less than that of Cl, must migrate behind the Cl and ahead of the glycine. o This has the effect of concentrating the proteins in a thin band sandwiched between the Cl ions and the glycine molecules. o All of the proteins in the gel sample have an electrophoretic mobility that is intermediate between the extreme of the mobility of the glycine and Cl- . o In addition, because the acrylamide concentration of the stacking gel is very low most proteins are not retarded and move freely through the gel matrix.
- 21. 4. This procession carries on until it hits the running gel, where the pH switches to 8.8. At this pH the glycine molecules are mostly negatively charged and can migrate much faster than the proteins. So the glycine front accelerates past the proteins, leaving them behind. Since the running gel has an increased acrylamide concentration, which slows the movement of the proteins according to their size, the separation begins When the sample reaches the end of the stacking gel, blue bands are found.
- 22. The end result of SDS-PAGE has two important features: All proteins contain only primary structure All proteins have a large negative charge which means they will all migrate towards the positive pole when placed in an electric field.
- 23. APPLICATIONS Determine protein size Identify protein Determine sample purity Identify existence of disulfide bonds Quantify amounts of protein
- 24. CLONING
- 25. Vector: A DNA molecule, capable of replication in a host organism, into which a gene is inserted to construct a recombinant DNA molecule. Shuttle vector: A vector that can replicate in the cells of more than one organism ExpressionVectors: This vector is designed specifically to promote expression of the cloned gene in a bacterial host.
- 26. Role of Isopropyl β-D-1- thiogalactopyranoside (IPTG) It is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon. IPTG binds to the lac repressor and releases the tetrameric repressor from the lac operator in an allosteric manner, thereby allowing the transcription of genes in the lac operon, such as the gene coding for beta-galactosidase, a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides