The BCA Protein Assay uses bicinchoninic acid to detect and quantify the amount of protein in a sample. It involves reducing Cu+2 to Cu+1 in an alkaline environment using proteins, which then complexes with BCA to produce a purple color. The absorbance at 562 nm is proportional to the protein concentration in the sample. It is highly sensitive, selective, and simpler than other assays like Lowry, though the color produced is not stable over time.

1. Protein Estimation and Detection

2. BCA (Bicinchoninic Acid) Protein Assay
Principle:
• Proteins 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
 Western 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:
i. Separation by size
ii. Transfer to a solid support
iii. Marking 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