Western blotting


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Western Blotting

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Western blotting

  1. 1. PRIYANKA ANJALI (Medico) Department of MicrobiologySri Dev Raj Urs Medical College, Kolar - Karnataka Presented by Priyanka Anjali, Designed by 1 Dr.T.V.Rao MD
  2. 2. What is blotting?Blots are techniques for transferring DNA , RNA andproteins onto a carrier so they can be separated, and oftenfollows the use of a gel electrophoresis. The Southern blotis used for transferring DNA, the Northern blot for RNAand the western blot for PROTEIN. Presented by Priyanka Anjali, Designed by 2 Dr.T.V.Rao MD
  3. 3. TYPES OF BLOTTING TECHNIQUES Blotting technique Southern Blot Northern Blot Western blotIt is used to detect DNA. It is used to detect RNA. It is used to detect protein.
  4. 4. Protein blottingProtein blotting is ananalytical method thatinvolves the immobilizationof proteins on membranesbefore detection usingmonoclonal or polyclonalantibodies. There aredifferent blotting protocols(dot blot, 2D blot); one of themost powerful is westernblotting. Presented by Priyanka Anjali, Designed by 4 Dr.T.V.Rao MD
  5. 5. Principle of Western BlottingWestern blotting is an Immunoblotting technique whichrely on the specificity of binding between a molecule ofinterest and a probe to allow detection of the molecule ofinterest in a mixture of many other similar molecules. In Western blotting, the molecule of interest is a proteinand the probe is typically an antibody raised against thatparticular protein.The SDS PAGE technique is a prerequisite for Westernblotting . Presented by Priyanka Anjali, Designed by 5 Dr.T.V.Rao MD
  6. 6. Western blot (Immunoblotting)A technique for detectingspecific proteinsseparated byelectrophoresis by use oflabeled antibodies. Socalled since it has somesimilarity to a Southernblot. Presented by Priyanka Anjali, Designed by 6 Dr.T.V.Rao MD
  7. 7. DefinitionThe Western Blot isan analyticaltechnique used todetect specificproteins in a givensample of tissuehomogenate orextract. Presented by Priyanka Anjali, Designed by 7 Dr.T.V.Rao MD
  8. 8. Advantages of Western BlotWestern blot analysis cananalyze any protein samplewhether from cells ortissues, but also can analyzerecombinant proteinssynthesized in vitro.Western blot is dependenton the quality of antibodyyou use to probe for yourprotein of interest, and howspecific it is for this protein Presented by Priyanka Anjali, Designed by 8 Dr.T.V.Rao MD
  9. 9. Gel electrophoresisUses gel electrophoresis toseparate native or denaturedproteins by the length of thepolypeptide (denaturingconditions) or by the 3-Dstructure of the protein(native/ non-denaturingconditions). The proteins arethen transferred to a membrane(typically nitrocellulose orPVDF), where they are probed(detected) using antibodiesspecific to the target protein. Presented by Priyanka Anjali, Designed by 9 Dr.T.V.Rao MD
  10. 10. Monoclonal and Polyclonal Antibodies are usedHere are now manyreagent companies thatspecialize in providingantibodies (bothmonoclonal andpolyclonal antibodies)against tens of thousandsof different proteins. Presented by Priyanka Anjali, Designed by 10 Dr.T.V.Rao MD
  11. 11. Most useful in ….This method is usedin the fields ofmolecular biology,biochemistry,immunogenetics andother molecularbiology disciplines. Presented by Priyanka Anjali, Designed by 11 Dr.T.V.Rao MD
  12. 12. The procedure includes..Tissue preparationGel electrophoresisTransferBlockingDetectionAnalysis Presented by Priyanka Anjali, Designed by 12 Dr.T.V.Rao MD
  13. 13. Tissue PreparationsSamples may be taken from whole tissue or from cell culture.In most cases, solid tissues are first broken downmechanically using a blender. It should be noted that bacteria, virus or environmentalsamples can be the source of protein and thus Westernblotting is not restricted to cellular studies only. Assorted detergents, salts, and buffers may be employed toencourage lysis of cells and to solubilize proteins. Tissue preparation is often done at cold temperatures toavoid protein denaturing. Presented by Priyanka Anjali, Designed by 13 Dr.T.V.Rao MD
  14. 14. Gel ElectrophoresisThe proteins of the sample are separated using gelelectrophoresis. Separation of proteins may be byisoelectric point molecular weight, electric charge, or acombination of these factors.The principle involved is the difference in theELECTROPHORETIC MOBILITIES of different proteins. Presented by Priyanka Anjali, Designed by 14 Dr.T.V.Rao MD
  15. 15. SDS-PAGE sample. Processing Presented by Priyanka Anjali, Designed by 15 Dr.T.V.Rao MD
  16. 16. Presented by Priyanka Anjali, Designed by 16Gel electrophoresis machine Dr.T.V.Rao MD
  17. 17. TransferringIn order to make the proteins accessible to antibody detection, theyare moved from within the gel onto a membrane made ofnitrocellulose or polyvinylidene difluoride (PVDF). The membraneis placed on top of the gel, and a stack of filter papers placed on topof that. The entire stack is placed in a buffer solution which movesup the paper by capillary action, bringing the proteins with it.Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gelinto the PVDF or nitrocellulose membrane. Presented by Priyanka Anjali, Designed by 17 Dr.T.V.Rao MD
  18. 18. BlockingThe membrane has the ability to bind to proteins in in thiscase both the target and antibodies are proteins and so therecould be some unwanted binding. Blocking of non-specific binding is achieved by placing themembrane in a dilute solution of protein - typically Bovineserum albumin(BSA) with a minute percentage of detergentsuch as Tween 20. The protein in the dilute solution attaches to the membranein all places where the target proteins have not attached.Thus, when the antibody is added, there is no room on themembrane for it to attach other than on the binding sites ofthe specific target protein. Presented by Priyanka Anjali, Designed by 18 Dr.T.V.Rao MD
  19. 19. DetectionDuring the detectionprocess, the membrane is"probed" for the protein ofinterest with a modifiedantibody which is linked toa reporter enzyme, whichwhen exposed to anappropriate substrate drivesa colorimetric reaction andproduces a color. Presented by Priyanka Anjali, Designed by 19 Dr.T.V.Rao MD
  20. 20. Presented by Priyanka Anjali, Designed by 20 Dr.T.V.Rao MD
  21. 21. AnalysisAfter the unbound probes are washed away, the westernblot is ready for detection of the probes that are labeledand bound to the protein of interest. Size approximations are taken by comparing the stainedbands to that of the marker loaded duringelectrophoresis. The process is repeated for a structural protein, such asactin or tubulin that should not change between samples. Presented by Priyanka Anjali, Designed by 21 Dr.T.V.Rao MD
  22. 22. AdvantagesWhile ELISA being a non specific test, Western blotting is a more specific test for detection of HIV.It can detect one protein in a mixture of proteins while giving information about the size of the protein and so is more specific.Western blot test is referred to as the Gold StandardIt also tells you how much protein has accumulated in cells. Presented by Priyanka Anjali, Designed by 22 Dr.T.V.Rao MD
  23. 23. Western Blot in Clinical MedicineThe confirmatory HIV test employs a Western blot to detect anti-HIVantibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane then, theserum to be tested is applied in the primary antibody incubation step;free antibody is washed away, and a secondary anti-human antibodylinked to an enzyme signal is added. The stained bands then indicatethe proteins to which the patients serum contains antibody.A Western blot is also used as the definitive test for Bovinespongiform encephalopathy (BSE, commonly referred to as mad cowdisease).Some forms of Lyme disease testing employ Western blotting. Presented by Priyanka Anjali, Designed by 23 Dr.T.V.Rao MD
  24. 24. Western Blot a Confirmatory test in HIV InfectionThe virus is envelopedwith different proteins.The detection of theseproteins are useful in thedetection of the presenceof the virus.Western blotting helps inthe detection of theseproteins. Presented by Priyanka Anjali, Designed by 24 Dr.T.V.Rao MD
  25. 25. DisadvantagesIf a protein is degradedquickly, Western blottingwont detect it well.This test takes longer thatother existing testsIt might also be morecostly Presented by Priyanka Anjali, Designed by 25 Dr.T.V.Rao MD
  26. 26. Programme presented by Miss Priyanka Anjali for e-learning Email doctortvrao@gmail.com Presented by Priyanka Anjali, Designed by 26 Dr.T.V.Rao MD