The bradford dye-binding assay is a colorimetric assay for measuring total protein concentration. It involves the binding of Coomassie Brilliant blue to protein.
There is no interference from cations nor from carbohydrates such as sucrose. However, detergents such as sodium dodecyl sulfate and triton x-100 can interfere with the assay, as well as strongly alkaline solutions.
*The following instructions are given for use with the BioRad model 2550 EIA reader in room 4.278
General Overview of Procedure:
Preparation of Standard
A set of standards is created from a stock of protein whose concentration is known. The Bradford values obtained for the standard are then used to construct a standard curve to which the unknown values obtained can be compared to determine their concentration. Use a protein as your standard that most closely resembles the protein you are assaying. BSA and IgG are typical standards used to construct the curve. For BSA, use 0-1 mg/mL as your standard curve concentration; for IgG, use 0-1.6 mg/mL.
Example of typical standards created from BSA stock (1 mg/mL), to give a standard curve from 0-1 mg/mL:
Sample #1 (0.0 mg/mL): 0 uL BSA + 30 uL buffer.
Sample #2 (0.2 mg/mL): 6 uL BSA + 24 uL buffer.
Sample #3 (0.4 mg/mL): 12 uL BSA + 18 uL buffer.
Sample #4 (0.6 mg/mL): 18 uL BSA + 12 uL buffer.
Sample #5 (0.8 mg/mL): 24 uL BSA + 6 uL buffer.
Sample #6 (1.0 mg/mL): 30 uL BSA + 0 uL buffeer.
*Note: The ubiquitously used BSA is a very poor standard for the bradford assay. To adjust for this, multiply the results you get for your protein concentration by 2.1 to get a closer approximation of your protein's concentration. Lysozyme, ovalbumin and catalase make much better standards, and no adjustment is necessary for these standards.
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