Bca Protein Assay Calculation

BCA Protein Assay Formula:

\[ \text{Conc (µg/mL)} = \frac{\text{Abs sample} - \text{Abs blank}}{\text{Slope}} \]

Abs Sample (absorbance):

Abs

Abs Blank (absorbance):

Abs

Slope (absorbance per µg/mL):

Abs/(µg/mL)

Concentration (µg/mL):

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1. What is the BCA Protein Assay?

The Bicinchoninic Acid (BCA) Protein Assay is a biochemical method for determining protein concentration. It's based on the reduction of Cu²⁺ to Cu⁺ by proteins in an alkaline medium, followed by colorimetric detection using BCA.

2. How Does the Calculation Work?

The calculation uses the standard curve equation:

\[ \text{Conc (µg/mL)} = \frac{\text{Abs sample} - \text{Abs blank}}{\text{Slope}} \]

Where:

\( \text{Abs sample} \) — Absorbance of the protein sample
\( \text{Abs blank} \) — Absorbance of the blank/reference
\( \text{Slope} \) — Slope from the standard curve (absorbance per µg/mL)

Explanation: The equation converts absorbance readings to protein concentration using the linear relationship established by the standard curve.

3. Importance of Protein Quantification

Details: Accurate protein quantification is essential for various biological and biochemical applications, including enzyme kinetics studies, protein purification, and Western blot normalization.

4. Using the Calculator

Tips: Enter absorbance values for both sample and blank, and the slope from your standard curve. Ensure all values are positive and the slope is greater than zero.

5. Frequently Asked Questions (FAQ)

Q1: Why subtract the blank absorbance?
A: The blank accounts for background absorbance from reagents and is subtracted to obtain the net absorbance due to protein.

Q2: How is the slope determined?
A: The slope is derived from a standard curve created by measuring absorbance of known protein concentrations (typically BSA standards).

Q3: What is the typical range for BCA assay?
A: The standard BCA assay is sensitive in the range of 20-2000 µg/mL, with microplate versions detecting 0.5-40 µg/mL.

Q4: What factors affect BCA assay results?
A: Reducing agents, certain detergents, and high salt concentrations can interfere with the assay. Sample composition should match standard buffer conditions.

Q5: How does BCA compare to Bradford assay?
A: BCA is generally more sensitive and less susceptible to interference from detergents, while Bradford is faster and less expensive.