BCA Protein Determination Calculator

BCA Protein Calculation:

\[ C = \frac{(A_{562} - blank)}{slope} \]

Protein Concentration (C):

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1. What is the BCA Protein Assay?

The Bicinchoninic Acid (BCA) assay is a colorimetric method for determining protein concentration. It is based on the reduction of Cu²⁺ to Cu⁺ by proteins in an alkaline medium, followed by color development through BCA chelation of Cu⁺.

2. How Does the Calculator Work?

The calculator uses the BCA protein determination formula:

\[ C = \frac{(A_{562} - blank)}{slope} \]

Where:

\( C \) — Protein concentration (μg/mL)
\( A_{562} \) — Sample absorbance at 562 nm
\( blank \) — Blank absorbance at 562 nm
\( slope \) — Slope from standard curve (absorbance/μg/mL)

Explanation: The equation converts absorbance readings to protein concentration using the standard curve generated from known protein standards.

3. Importance of Protein Quantification

Details: Accurate protein quantification is essential for experimental consistency, sample normalization, and ensuring proper protein loading in downstream applications like Western blotting or enzymatic assays.

4. Using the Calculator

Tips: Enter the sample absorbance (A562), blank absorbance, and standard curve slope. All values must be valid (slope ≠ 0). For best results, use the standard curve generated from the same experiment.

5. Frequently Asked Questions (FAQ)

Q1: What is the typical range for BCA assays?
A: The BCA assay is sensitive from 20-2000 μg/mL, though this depends on the specific protocol and standard curve.

Q2: How should I prepare my standard curve?
A: Use a series of known protein concentrations (typically BSA) covering your expected sample range, processed identically to your samples.

Q3: What affects BCA assay accuracy?
A: Factors include protein composition (different proteins may reduce Cu²⁺ differently), interfering substances (e.g., reducing agents), and incubation time/temperature.

Q4: Should I use the same blank for all samples?
A: Yes, use the same blank measurement for all calculations to ensure consistency.

Q5: How does BCA compare to Bradford assay?
A: BCA is generally more sensitive and less affected by detergents, but Bradford is faster and may be better for certain protein types.