Protein Concentration Calculator Bradford Assay

Bradford Assay Formula:

\[ \text{Conc (µg/mL)} = \frac{\text{Abs}}{\text{Standard curve slope}} \]

Protein Concentration:

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1. What is the Bradford Assay?

The Bradford assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. It is based on the shift in absorbance maximum of Coomassie Brilliant Blue G-250 dye from 465 nm to 595 nm when binding to proteins.

2. How Does the Calculator Work?

The calculator uses the Bradford assay formula:

\[ \text{Conc (µg/mL)} = \frac{\text{Abs}}{\text{Standard curve slope}} \]

Where:

\( Abs \) — Absorbance reading at 595 nm
\( Slope \) — Slope of the standard curve (AU/(µg/mL))

Explanation: The calculation assumes a linear relationship between absorbance and protein concentration, which is valid within the assay's dynamic range.

3. Importance of Protein Quantification

Details: Accurate protein quantification is essential for various biochemical applications including enzyme kinetics, Western blotting, protein purification, and clinical diagnostics.

4. Using the Calculator

Tips: Enter the absorbance reading from your sample and the slope determined from your protein standard curve. Ensure measurements are within the linear range of the assay (typically 1-1500 µg/mL for BSA).

5. Frequently Asked Questions (FAQ)

Q1: What is the typical range for Bradford assay?
A: The Bradford assay typically has a working range of 1-1500 µg/mL when using bovine serum albumin (BSA) as a standard.

Q2: Why do I need a standard curve?
A: A standard curve is necessary to establish the relationship between absorbance and protein concentration, as different proteins may yield different color responses.

Q3: How do I create a standard curve?
A: Prepare a series of known protein concentrations, measure their absorbance, and plot absorbance vs. concentration. The slope of this line is used in the calculation.

Q4: Are there limitations to the Bradford assay?
A: Yes, the assay can be affected by detergents, certain buffers, and different proteins may yield different color responses (variable dye binding).

Q5: What wavelength should I use for measurements?
A: Absorbance should be measured at 595 nm, which is the absorbance maximum for the protein-dye complex.