In order to determine the protein concentration of an unknown sample using bradford reagent, I have used BSA with concentration of 1mg/ml as standard. to plot a standard curve I have used 2,4,6,8 and 10 ul of BSA. What volume of unknown sample should I use to measure the absorbance? What would be the unit of concentration after solving the equation from standard curve? Does the volume of sample used affect the answer?
If you want to use Bradford's method, do not use BSA as standard, as this protein has hydrophobic binding pockets, which bind more dye than an "average" protein. Immunoglobulin works better for most samples.
You compare the slope of the absorbance as function of volume of the standard line with that of your sample, and then calculate the unknown concentration from both slopes and the known concentration by the rule of three. The absorbances obtained with the sample should be of similar magnitude as those obtained with the standard.
As an additional problem, the data points with Bradford's method can be approximated by a straight line only for a small concentration range, for larger ranges, the data form curves that are best approximated by a quadratic equation.
To simplify interpretation of the protein assay (Bradford or others), use units of micrograms of protein, rather than concentrations. The standard curve you made would then be 0, 2, 4, 6, 8, 10 µg of BSA.
The unknown sample volume should be identical to the BSA standard volume, and all the BSA standards should have the same volume. This requires that various dilutions of the BSA stock solution be made, of course.
For example, I perform the Bradford assay in a 96-well plate format, using a 10 µl volume of sample or standard and 200 µl of Bradford reagent. I prepare BSA standards in distilled water such that 10 µl contains 0, 1, 2, 3, etc. µg of BSA.
The volume of the unknown will also be 10 µl. It will consist of some volume of the sample + water to bring the volume to 10 µl. (Note: if there is anything in the sample that interferes with the assay, the amount of the interfering substance must be the same in the standards and sample.) A little qualitative experimentation with the sample before starting the assay helps to choose the right amount to add, or you can test several amounts at once to make sure you get at least one test that is within the standard curve.
Suppose I use 3 µl of the sample and find that it had 4 .5 µg of protein based on the standard curve. Then the concentration is 4.5 µg/3 µl = 1.5 mg/ml.
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