BCA and Western blot (1).pptx

Basics of western blot

1. Protein Estimation and Detection

2. BCA (Bicinchoninic Acid) Protein Assay Principle: P roteins can reduce Cu +2  to Cu +1  in an alkaline solution (the biuret reaction) and result in a purple color formation by bicinchoninic acid The reaction between a protein and the BCA reagent form a complex that absorbs light at 562 nm. A protein quantification assay used to determine the concentration of proteins in a sample. The absorbance is proportional to the concentration of protein in the sample. Highly sensitive and selective colorimetric detection of the cuprous cation (Cu+) by BCA .

3. Materials Materials provided in the kit: BCA reagent A BCA reagent B Materials required, but not provided in the kit: Tubes Spectrophotometer

4. Procedure

5. Analysis of the Result 562 nm BSA Standard Calibration Curve

6. Advantages: As sensitive as Lowry but simpler. Reagents more stable than Lowry. Disadvantages: Color not stable with time… Precise timing. Color variations between proteins occur. Advantages and Disadvantages of BCA Method

7. Western Blotting W estern blotting is a widely used analytic technique in molecular biology and immunogenetics to detect specific proteins from a complex mixture of proteins extracted from cells. Was first described on Towbin , et. al in 1979. The SDS PAGE technique is prerequisite for Western blotting. Proteins are separated based on their size on polyacrylamide gel. The technique uses three elements to accomplish this task: Separation by size T ransfer to a solid support M arking target protein using a proper primary and secondary antibody to visualize . Very delicate and time consuming process.

8. Sample Preparation Sample

9. Western Blot Setup

10. Procedure

11. Advantages and Limitations of Western Blot Advantages of Western Blot: Specific interaction of antibody and antigen can be directly visualized. Analytical tool to identify a protein of interest in a complex mixture. To identify molecular weight of the protein. Widely accepted method. Limitations of Western Blot: Very delicate and time consuming process. If protein degrade quickly, western blot wont detect it well. Technically demanding. Costly.

12. Immunogenic responses from infectious agents (ex. viruses, bacteria) are easy to detect by this technique. Analysis of IgG fractions purifies from human plasma. Diagnosis of HIV by ELISA involves western blotting technique. To detect some forms Lyme disease. Confirmatory test for hepatitis-B involves western blotting technique. This technique employed in gene expression studies. Applications of Western Blot