Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
|
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). |
|||||||||||||
| Solutions |
|||||||||||||
|
|||||||||||||
| BioReagents and Chemicals |
|||||||||||||
|
Bovine Serum Albumin Coomassie Protein Assay Reagent Protein Assay Reagent |
|||||||||||||
| Protocol Hints |
|||||||||||||
|
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). |
|||||||||||||
