Western Blot Medical Presentation

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Western Blot Prepared by : Yousra Esmat Under supervision of : Dr. Radwa Mahanha
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INTRODUCTION Western blotting (WB) is a popular laboratory technique to detect specific proteins from a cell or tissue sample. The technique was initially described by Towbin et al. in 1979.
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PRINCIPLE WB requires to separate proteins according to their size by gel electrophoresis. It is then followed by electrotransfer of the proteins to a membrane that is probed with an antibody against the protein of interest.
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Flowchart of Western Blot Procedure
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I- Sample Preparation
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1- Tissue Homogenization Tissue homogenization (mechanical or enzymatic) to extract and solubilize proteins.
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2- Cell Lysis Lysis buffer which is composed of: Buffering agent (e.g., Tris–HCl), Salts such as sodium chloride, Detergent disrupting lipids, Protease inhibitor.
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3- Lowry Assay Protein concentration in the lysates can be measured using Lowry assays to: This ensures equal loading of proteins from different samples Allow comparison between experiments.
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4- Loading Buffer Mix the samples with a loading buffer composed of : Glycerol to facilitate sample loading on the gel, Coomassie Blue dye to see the sample β-mercaptoethanol to reduce disulfide bonds Sodium dodecyl sulphate (SDS) in case of denaturing conditions.
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II- Electrophoresis
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Protein Separation Samples are then subjected to polyacrylamide gel electrophoresis (PAGE), which separates proteins either based on : Isoelectric point (native-PAGE) or Size (SDS-PAGE). Proteins need to possess a negative charge to migrate through the gel pores when subjected to an electromagnetic field.
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Polyacrylamide gel It is a mix of polyacrylamide and bis-acrylamide, which form a crosslinked polymer by the polymerizing agent ammonium persulfate (APS) Gel pore size is regulated by the ratio and concentration of polyacrylamide and bis-polyacrylamide and needs to be adjusted to the size of the protein of interest. Higher concentrations and increased amount of bis-acrylamide give smaller pores separating smaller proteins and vice versa.
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- Lower % acrylamide and pH, - Different ionic composition to “compress” proteins.
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Schematic of SDS-PAGE Electrophoresis
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III- Electrotransfer Proteins separated by PAGE need to be transferred from the gel to a membrane by electrophoretic transfer for further processing.
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Principle Membrane is tightly touching the gel and sandwiched between filter paper in a transfer buffer. (avoid air bubbles) Electromagnetic field applied perpendicularly to the gel to transfer the proteins from the gel to the membrane.
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IV. Blocking
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Blocking Membranes used in electrotransfer are characterized by high protein binding capabilities, thus surface unoccupied by transferred proteins requires blocking to reduce background. Ideal blocking solutions: Nonfat dry milk (NFDM) Bovine serum albumin (BSA).
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V. Probing This step is crucial and may require extensive optimization as each antibody will have different strength and specificity.
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Washings Usually a very mild detergent solution is used for washings. Unbound antibodies should be removed without affecting the specific binding of the antibody to its target ( increases background). A balance should be achieved between removal of the background and ability to detect the specific signal.
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VI. Detection
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Substrate releases light Detected by X-ray films Substrate gives a visible end-product precipitated on the membrane which is monitored in real-time. less sensitive Less sensitive. Better quantification. Several targets in a single run.
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APPLICATIONS
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Research applications 1- Determination of the presence of a protein of interest. 2- Determination of changes in protein expression levels (when samples are collected at different time points). 3- Assessment of posttranslational modification of the target protein such as phosphorylation (with phospho-specific antibodies).
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Diagnostic applications WB is used to help in diagnosis of some diseases where high degree of confidence is necessary. In medicine, it is often used as a confirmatory test [with ELISA] in diagnosis of several diseases such as lyme disease or HIV. WB is used to detect the prion protein causing bovine spongiform encephalopathy, bovine spongiform encephalopathy (BSE) in cows.
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LIMITATIONS
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1- Quantification WB is not a quantitative technique as it will not tell how much of the protein is expressed. Only relative differences in expression levels can be assessed and even this needs to be approached with caution. Imaging with charge coupled device (CCD) cameras sensitive to chemiluminescence is much better for quantification purposes but is less sensitive.
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2- Sensitivity WB in general is quite sensitive, however, due to accumulation of intrinsic inefficiencies of the technique (such as transfer, recognition by antibodies) the detection limit is usually higher. A negative result cannot be interpreted as complete lack of the target protein in the sample (not all expressed proteins can be detected).
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3- Specificity WB is as specific as the antibody used for the membrane probing when the specificity of the antibody is poor the result may be not easy to interpret.
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TROUBLESHOOTING
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REFERENCES 1- Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979;76(9):4350. [2] Burnette WN. “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate – polyacrylamide gels to unmodifed nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 1981;112(2):195–203
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REFERENCES 3- Soundy P, Harvey B. Western blotting as a diagnostic method Medical biomethods handbook. Springer; 2005:43–62. 4- Schaller O, Fatzer R, Stack M, Clark J, Cooley W, Biffger K, et al. Validation of a Western immunoblotting procedure for bovine PrPSc detection and its use as a rapid surveillance method for the diagnosis of bovine spongiform encephalopathy (BSE). Acta Neuropathol Springer 1999;98(5):437–43
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