Basics:

• Principle: The BCA assay is based on the reduction of copper ions (Cu^2+) to copper ions (Cu^+) by protein in an alkaline medium. The resulting Cu^+ ion then forms a colored complex with Bicinchoninic Acid, which can be measured spectrophotometrically.
• Absorbance: The intensity of the color produced is proportional to the amount of protein in the sample. The absorbance of this complex is typically measured at 562 nm.

Components:

1. Protein sample: Could be from cell lysates, tissue extracts, or purified proteins.
2. BCA reagent: Contains bicinchoninic acid and copper sulfate. This reagent reacts with the proteins in the sample to produce the colored complex.
3. Standard: Bovine Serum Albumin (BSA) is often used as a standard to generate a calibration curve, hence the name “BSA assay”. However, other proteins can also be used as standards.

Procedure:

1. Sample Preparation: Protein samples are typically diluted in appropriate buffers to fall within the detectable range of the assay.
2. Reaction Setup: Protein samples and BSA standards are mixed with the BCA reagent.
3. Incubation: The mixtures are incubated, typically at 37°C for 30 minutes, though this can vary.
4. Measurement: The absorbance of each sample is measured at 562 nm using a spectrophotometer or a plate reader.
5. Quantification: By comparing the absorbance values of the samples to the BSA standard curve, the concentration of protein in the unknown samples can be determined.

Advantages:

• Relatively simple and quick.
• More tolerant to the presence of detergents and other substances compared to some other protein assays (like the Bradford assay).

Limitations:

• Some compounds (e.g., chelating agents, strong reducing agents) can interfere with the assay.
• Can sometimes be less sensitive than other assays, such as the Lowry or Bradford assays.

Applications:

• Determining protein concentration in cell lysates before SDS-PAGE or Western Blotting.
• Protein quantification in various biological and biochemical research applications.

When using the BCA assay, it’s essential to optimize and validate the assay for the specific conditions and samples you are working with to ensure accurate results.