Advanced techniques in protein estimation
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This document discusses various techniques for estimating proteins including immunohistochemistry, PCR, blotting techniques, SDS-PAGE, and spectroscopy. It provides details on each technique such as the principles, procedures, and applications. Immunohistochemistry uses antigen-antibody reactions to identify specific tissue components. PCR amplifies segments of DNA through repeated cycles of denaturation, annealing, and extension. Blotting techniques like Southern, Northern, and Western blotting are used to transfer and detect DNA, RNA, and proteins. SDS-PAGE separates proteins by size using polyacrylamide gel electrophoresis. Spectroscopy techniques like MALDI-TOF mass spectrometry are used to analyze proteins and identify disease markers.
Advanced techniques in protein estimation
- 1. Advance Techniques in Protein Estimation SINHGAD INSTITUTE OF PHARMACY, NARHE Presented by Guide Mr. Gogawale Vinayak G. M. Pharm. (Semester-III) (Department of Pharmacology) Dr. U. M. Aswar (Asst. Professor Pharmacology)
- 2. CONTENTS Introduction Techniques for protein estimation Immuno histochemistry PCR Blotting techniques SDS Page Spectroscopy 18-06-2016 2
- 3. Introduction • Proteins are the . 18-06-2016 3
- 4. Techniques for protein estimation • Immuno histochemistry • PCR • Blotting techniques • SDS Page • Spectroscopy 18-06-2016 4
- 5. Immuno histochemistry • Immunohistochemistry is the technique in which combination of histological, immunological and biochemical techniques for the identification of specific tissue components. • Antigen/antibody reaction labeled with a visible label. • Possible to identify any cell component within cell or tissue. 18-06-2016 5
- 6. Cont…. • Requirements for immuno-histochemistry : • Antibody • Antigen Retrieval • Blocking • Methods for IHC: • Direct method • Indirect method • Immunoenzyme • Fluorescence • Multiple labeling 18-06-2016 6
- 7. 18-06-2016 7 • Antibodies: • Immune cells, or B cells, produce antibodies against targeted proteins. • They have two types Monoclonal and polyclonal antibodies. • a monoclonal antibody preparation contains a single antibody with specificity to one epitope on the antigen molecule. • Polyclonal antibodies are different B cell produced antibodies against different sites on the same antigen.
- 8. 18-06-2016 8 Cont…. Method for Monoclonal antibodies
- 9. • Antigen Retrieval: Routine histology procedures masks the antigens, usually by formalin cross-linking, or even destroys some antigen epitopes. 18-06-2016 9
- 10. Blocking 18-06-2016 10 In principle, any protein that does not specifically bind to your epitope of interest, or to the antibodies in your immunohistochemistry assay, can be used to block. The samples are incubated with a buffer that blocks the reactive sites to which the primary or secondary antibodies may otherwise bind. Common blocking buffers include normal serum or BSA.
- 11. Methods for IHC Direct method Indirect method 18-06-2016 11
- 12. 18-06-2016 12
- 13. • It is method for amplifying particular segments of DNA, distinct from cloning and propagation within the host cell. • The procedure is carried out entirely biochemically, that is, in vitro. • PCR was invented by Kary Mullis in 1983. He shared the Nobel Prize in chemistry with Michael Smith in 1993. Polymerase Chain Reaction 18-06-2016 13
- 14. PRINCIPLE • PCR uses the enzyme DNA polymerase that directs the synthesis of DNA from deoxynucleotide substrates on a single-stranded DNA template. • DNA polymerase adds nucleotides to the 3` end of a custom-designed oligonucleotide when it is annealed to a longer template DNA. • DNA polymerase can use the oligonucleotide as a primer and elongate its 3` end to generate an extended region of double stranded DNA. 18-06-2016 14
- 15. PCR Steps: • Denaturation step (90–95 °C) This breaks the weak hydrogen bonds that hold DNA strands together in a helix It allowing the strands to separate creating single stranded DNA. • Annealing step (50–65 °C) This allows the primers to bind (anneal) to their complementary sequence in the template DNA. Cont…. 18-06-2016 15
- 16. • Extension/Elongation step (up to 72° C) DNA polymerase extends the primers, adding nucleotides onto the primer in a sequential manner, using the target DNA as a template. With one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double- stranded DNA. These two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the template is increased exponentially. Cont…. 18-06-2016 16
- 18. Blotting Techniques Southern blotting • Used for transferring DNA • Hybridization • Nitrocellulose filter membrane Northern blotting • Used for transferring RNA • Hybridization • Amonobenzyloxy methyl filter paper Western blotting • Used for transferring Proteins • Nitrocellulose filter membrane 18-06-2016 18
- 19. Southern Blotting • Southern blot is a method used to check for the presence of a DNA sequence in a DNA sample. • Hybridization of proteins is the main process. • The principle of hybridization analysis is that a single-stranded DNA or RNA molecule of defined sequence (the probe) can base-pair to a second DNA or RNA molecule that contains a complementary sequence (the target), with the stability of the hybrid depending on the extent of base pairing that occurs.18-06-2016 19
- 20. 18-06-2016 20 Cont…. Procedure for Southern Blot
- 21. Northern Blot • The northern blot technique is used to study gene expression by detection of RNA (or isolated mRNA) in a sample. • With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions. 18-06-2016 21
- 22. 18-06-2016 22 Cont…. Procedure for blot
- 23. Western Blotting • Western blotting is an Immunoblotting technique which rely on the specificity of binding between a molecule of interest and a probe to allow detection of the molecule of interest in a mixture of many other similar molecules. • It is dependent on the quality of antibody use for probe to protein of interest, and how specific it is for this protein 18-06-2016 23
- 24. Steps involved in western blotting • Tissue preparation • Gel electrophoresis • SDS PAGE • Transfer • Blocking • Detection • Analysis18-06-2016 24 Cont….
- 25. SDS PAGE • Sodium dodecyl sulfate poly acrylamide gel electrophoresis. • Poly acrylamide gel as a support medium and sodium dodecyl sulphate (SDS) to denature the proteins. The method is called sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS-PAGE). • The purpose of SDSPAGE is to separate proteins according to their size. 18-06-2016 25
- 26. • SDS is an anionic detergent, meaning that when dissolved its molecules have a net negative charge within a wide pH range. A polypeptide chain binds amounts of SDS in proportion to its relative molecular mass. The negative charges on SDS destroy most of the complex structure of proteins, and are strongly attracted toward an anode (positively-charged electrode) in an electric field. 18-06-2016 26 Cont….
- 27. • The final separation of proteins is dependent almost entirely on the differences in relative molecular mass of polypeptides. • If proteins of known mass are run simultaneously with the unknowns, the relationship between Rf (distance travelled by proteins) and mass can be plotted, and the masses of unknown proteins estimated. 18-06-2016 27 Cont….
- 28. Transferring • In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. • Another method for transferring the proteins is called electro blotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. 18-06-2016 28
- 29. Blocking • The membrane has the ability to bind to proteins in this case both the target and antibodies are proteins and so there could be some unwanted binding. • Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein - typically Bovine serum albumin with a minute percentage of detergent such as Tween 20. The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. • Thus, when the antibody is added, there is no space on the membrane for it to attach other than on the binding sites of the specific target protein.18-06-2016 29
- 30. Detection 18-06-2016 30 •During the detection process, the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzyme, which when exposed to an appropriate substrate drives a colorimetric reaction and produces a color.
- 31. 18-06-2016 31 Cont…. Application of western blot
- 32. MALDI- TOFMS • Matrix Assisted Laser Desorption/Ionization- Time of Flight Mass Spectrometry. • The sample for MALDI is uniformly mixed in a large quantity of matrix. • The matrix absorbs the ultraviolet light (nitrogen laser light, wavelength 337 nm) and converts it to heat energy. • A small part of the matrix heats rapidly (in several nano seconds) and is vaporized, together with the sample. 18-06-2016 32
- 34. • MALDI-Tof-MS for identification of protein disease markers • It has been developed for use in clinical chemistry as a primary investigative tool to characterize cancer, Alzheimer, arthritis, and allergy protein markers of disease or susceptibility to disease. APPLICATIONS 18-06-2016 34
- 36. References • Aragno M., Mastrocola R., Alloatti G., Vercellinatto I., Bardini P., Geuna S., Catalano M. G., Danni O., Boccuzzi G., “Oxidative stress triggers cardiac fibrosis in the heart Of diabetic rats”, Endocrinology (2007), 149(1), 380–388. • Tzeng T. F., Liou S. S., Chang C. J., Liu I. M., “The ethanol extract of zingiber zerumbet attenuates streptozotocin-induced diabetic nephropathy in rats” ,Evidence-Based Complementary and Alternative Medicine(2013),1-8. • Valones M. A., Guimarães R. L., Branado L. A., De Souza P. R., “Principles and applications of polymerase chain reaction in medical Diagnostic fields: a review”, Brazilian Journal of Microbiology (2009) 40:1-11. 18-06-2016 36
- 37. • Marvin L. F., Roberts M. A., Flay L. B., “Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in clinical chemistry”, Clinica Chimica Acta., (2003).,337, 11 –21. • Liu Y. S., Huang Z. W., Wang L., Liu X. X., “Sitagliptin alleviated myocardial remodeling of the left ventricle and improved cardiac diastolic dysfunction in diabetic rats”, Journal of Pharmacological Sciences (2015), 127, 260-274. 18-06-2016 37 Cont….