Hello together,
I am trying to establish the protein-assy by Bradford (1976) following the instructions from "The Protein Protocols Handbook" edited by J.M. Walker. I am struggling with the measuerment of protein standard and kalibration curve. Hopefully, one of you can give an answer.
The chapter from Dr. N.J. Kruger describes, that the protein-standard should have an OD of 1.35 at 280 nm when gamma-globulin (1 mg/ml) is used. Every time I solve my frozen crystals of y-globulin (50 mg) and in 50 ml distilled water, the measuered OD is ~ 1.01. So I add much more y-globulin to achive the needed OD of 1.35. Following, it is said in the chapter that 100 µl of this standard + 5 ml of the reagent should have an OD of 0.4 at 595 nm. I always measure an OD of 1.2. Do you know my misstake?
Many thanks.
Dear Astrid,
I think that you are worrying too much about the discrepancy between your data and the published protocol says. The usual way to determine a calibration curve is to perform the assay on a series of samples with known amounts of a specific protein (most people use BSA because it is cheap, but gamma-globulin is OK too). The chromogenic effect may depend on the protein and on the reagent. Hence, whatever the result you may obtain, it will be valid if you calculate the concentration of your sample based on YOUR standard curve and if you state in the report that the standard was based on gamma-globulin.
You may save on the reagent by scaling down the assay to 1 ml instead of 5 ml. It is best to use single-use disposable plastic cuvettes, since the reaction tends to leave a blue film on the walls of the cuvette, which may interfere with further measurements. It is also advisable not to saturate the ragent with too much protein: with the Bio-Rad reagent, I used to perform a standard curve with 0-15 µg BSA in a volume of 1 ml. Note also that the assay is non linear: it is thus not legitimate to perform a linear regression on the points of the standard curve, nor to extrapolate for values that lie beyond the range of the standard curve.
Hello Pierre,
many thanks for your advice. I will proceed following your recommendations.
Actually, the use of Ig as standard for Bradford's assay is correct, BSA has hydrophobic pockets that bind more dye than "normal" proteins. Thus you would systematically underestimate the protein concentration of your sample.
If you want to use regression, fit the standard curve as a second order polynom (y = a + bx + cx2) to take care of the non-linearity.
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