WESTERN BLOTTING - immunology and immunotechnology

This presentation outlines the methodology of Western blotting, a powerful tool for protein detection and analysis, and its applications in research and diagnostics.

1. WESTERN BLOTTING PRESENTED BY HARIPRIYA U VARMA 2 ND MSC BIOTECHNOLOGY

2. INTRODUCTION Western blotting is a powerful tool for protein analysis, also known as immunoblotting, is a widely used technique in molecular biology for detecting specific proteins in complex mixtures. It combines gel electrophoresis, protein transfer, and antibody-based detection to provide a sensitive and quantitative method for studying protein expression, modifications, and interactions. The immunodetection of proteins has a wide application in biochemistry and other sciences as it can detect and characterize a multitude of proteins. Western blotting has a number of advantages over other similar techniques as the process only requires the use of a small amount of reagents, and the same protein transfer can be used for multiple analyses. 

3. PRINCIPLES OF WESTERN BLOTTING 1. Protein Separation Proteins are separated based on their molecular weight through gel electrophoresis, where smaller proteins migrate faster than larger ones. 2 . Protein Transfer Separated proteins are transferred from the gel onto a membrane, where they can be probed with antibodies. 3 . Antibody Binding Specific antibodies bind to the target protein on the membrane, allowing for its detection and quantification. 4. Signal Detection A secondary antibody coupled to an enzyme or fluorescent dye allows for visualization of the bound primary antibody and the target protein.

4. SAMPLE PREPARATION 1. Cell Lysis Cells are lysed to release proteins from their cellular environment using appropriate lysis buffers. 2. Protein Quantification Protein concentration is determined to ensure equal loading of samples onto the gel. 3. Sample Preparation Samples are prepared for electrophoresis, usually by mixing with a sample buffer containing SDS and reducing agents.

5. GEL ELECTROPHORESIS 1. Gel Preparation An appropriate polyacrylamide gel is prepared, with pore sizes that allow for effective separation of proteins based on size. 2. Sample Loading Samples are loaded into wells at the top of the gel, along with molecular weight markers for reference. 3. Electrophoresis An electric current is applied to the gel, causing proteins to migrate through the gel matrix.

6. PROTEIN TRANSFER 1. Transfer Setup The gel is placed on a membrane in a transfer apparatus, along with blotting paper and sponges. 2. Electric Current An electric current is applied, driving proteins from the gel onto the membrane. 3. Transfer Completion Proteins are transferred to the membrane, where they can be probed with antibodies.

7. ANTIBODY INCUBATION 1. Blocking The membrane is blocked with a solution that prevents non-specific antibody binding, ensuring that antibodies bind only to the target protein. 2. Primary Antibody A primary antibody specific to the target protein is incubated with the membrane, allowing it to bind to the protein. 3. Secondary Antibody A secondary antibody, which recognizes the primary antibody, is incubated with the membrane, allowing for detection and visualization.

8. DETECTION AND VISUALIZATION 1. Chemiluminescence The secondary antibody is conjugated with an enzyme that produces light when a substrate is added, allowing for detection of the target protein. 2. Colorimetric The secondary antibody is conjugated with an enzyme that produces a colored product when a substrate is added, allowing for detection of the target protein. 3. Fluorescenc e The secondary antibody is conjugated with a fluorescent dye that emits light when excited with a specific wavelength, allowing for detection of the target protein.

9. DATA ANALYSIS 1. Quantification of Bands Use image analysis software to measure the intensity of protein bands on the blot, ensuring to subtract background noise for accurate quantification. 2. Normalization Normalize the intensity of the target protein to a loading control (e.g., GAPDH) or total protein to account for variations in sample loading. 3. Data Interpretation Compare the normalized protein levels between different samples or conditions, followed by statistical analysis to assess the significance of observed differences.

10. TROUBLESHOOTING 1. No Signal Check for issues in sample preparation, antibody concentration, or blocking steps. 2. Non-Specific Bands Optimize blocking conditions or use higher affinity antibodies to reduce non-specific binding. 3. Weak Signal Increase antibody concentration or optimize detection conditions. 4. Smearing Ensure proper sample preparation, minimize heating during electrophoresis, and optimize transfer conditions .

11. APPLICATIONS To determine the size and amount of protein in given sample. Disease diagnosis: detects antibody against virus or bacteria in serum. Western blotting technique is the confirmatory test for HIV. It detects anti HIV antibody in patient’s serum. Useful to detect defective proteins. For eg Prions disease. Definitive test for Creutzfeldt-Jacob disease, Lyme disease, Hepatitis B and Herpes LIMITATIONS The test can only be used as a semi-quantitative test as the estimation is not always precise. The process is time-consuming and complex, thus can only be performed by well-trained personnel. Western blotting can only be performed for proteins if the primary antibodies for the proteins are available. The technique is a costly process with the cost of antibodies and expensive detection methods. Some antibodies might exhibit off-target effects by interacting with more than one protein in the sample.

12. CONCLUSION Western blotting is a powerful and versatile technique essential for detecting and quantifying specific proteins within complex biological samples. It combines precision in protein separation with the specificity of antibody detection, making it invaluable in research and diagnostics. Accurate data analysis, including proper quantification, normalization, and interpretation, ensures the reliability of results. Overall, Western blotting remains a cornerstone method in molecular biology for studying protein expression and function.