1. What is the BCA Assay?

The Bicinchoninic Acid (BCA) assay is a colorimetric method for quantifying total protein concentration. It's based on the reduction of Cu²⁺ to Cu⁺ by protein in an alkaline medium, followed by color development with BCA reagent.

2. How Does the Calculator Work?

The calculator uses the BCA assay formula:

Where:

• \( C \) — Protein concentration (μg/mL)
• \( A_{562} \) — Sample absorbance at 562 nm
• \( \text{blank} \) — Absorbance of blank/reagent control
• \( \text{slope} \) — Slope of standard curve (from BSA standards)

Explanation: The formula subtracts background absorbance (blank) and divides by the standard curve slope to determine protein concentration.

3. Importance of Protein Quantification

Details: Accurate protein quantification is essential for sample normalization, enzyme assays, Western blotting, and many other biochemical applications.

4. Using the Calculator

Tips:

• Measure absorbance at 562 nm (or nearest available wavelength)
• Use the same blank as used for standard curve generation
• Slope should be from a fresh standard curve (typically 0.001-0.1 μg/mL^-1)
• Sample absorbance should be within the linear range of the standard curve

5. Frequently Asked Questions (FAQ)

Q1: What's the typical range for BCA assays?
A: The working range is usually 20-2000 μg/mL, depending on protocol variations.

Q2: How do I determine the slope?
A: Create a standard curve with known BSA concentrations, measure their absorbance, and calculate slope from the linear regression.

Q3: Why subtract the blank?
A: The blank accounts for background absorbance from the reagents and any nonspecific color development.

Q4: What if my sample is outside the standard curve range?
A: Either dilute the sample (if too high) or concentrate it (if too low) and re-measure.

Q5: How does BCA compare to Bradford assay?
A: BCA is generally more sensitive and less affected by detergents, but more expensive and time-consuming.