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MOLECULAR BIOLOGY: WORKING WITH PROTEINS
DETERMINATION OF PROTEIN CONCENTRATION USING A MICROBRADFORD ASSAY AND MICROTITER PLATE READER
Determination of Protein Concentration Using a Microbradford Assay and Microtiter Plate Reader
Contributor: The Laboratory of Donald Rio at the University of California, Berkeley Procedure
1. Prepare a standard set of solutions with Bovine Serum Albumin ranging from 0.1 to 1 mg/ml in 0.1 mg/ml increments. Use the same solution for the protein samples (the assay is reported to be linear from 0.1 to 1 mg/ml protein).
2. Distribute 95 μl aliquots of the Protein Assay Reagent into the wells of a flat bottom microtiter plate.
3. Add EITHER 5 μl of the protein sample that you wish to analyze (in triplicate) or 5 μl of the Bovine Serum Albumin standard (in triplicate) to each well.
4. Mix each well by pipetting the solution up and down (see Hint #1).
5. Incubate at room temperature for 2 hours.
6. Determine the absorbance at 570 nm (A570) with a microtiter plate reader.
7. Calculate a linear regression curve (see Hint #2)
8. Calculate the concentration of unknown samples with the linear regression curve (by plotting along the x-axis the concentration in mg/ml of the protein standards).
Protein Assay Reagent Protein Assay Reagent available from Pierce Coomassie Protein Assay Reagent Coomassie Protein Assay Reagent available from Pierce BioReagents and Chemicals
Bovine Serum Albumin
Coomassie Protein Assay Reagent
Protein Assay Reagent
1. Do not introduce any bubbles into the solution.
2. Linear regression analysis: Plot along the X-axis the protein standard concentration (mg/ml) and along the Y-axis the A570. Calculate the best linear fit (y=mx+b) where y is mg/ml, m is the slope of the line, b is the y-intercept, and x is the absorbance value (A570).