I performed several Bradford assays to quantify proteins in human serum, but the absorbance of undiluted serum (with and without protease inhibitors) consistently showed negative values or values lower than the blank. Interestingly, diluted samples (1:5 and 1:10) gave higher absorbance than the undiluted serum.
In the assay, I added 20 µL of undiluted serum to 180 µL of 1× Bradford reagent (also tested with 10 µL of serum + 40 µL of 1× reagent), and a clear color change was visible upon reagent addition. Furthermore, SDS-PAGE analysis revealed well-defined protein bands.
What could be interfering with the Bradford reagent in human serum to cause this effect?
The proper way to perform the Bradford assay, or any protein assay, is to prepare a standard curve (serum albumin would be an appropriate standard in this case, since it is the most abundant protein in serum) to cover the dynamic range of the measurement. Various dilutions of the serum samples of unknown concentration should be prepared in the same buffer (or distilled water) as the standards. Only those dilutions that give absorbance values within the dynamic range of the standard curve should be used for the calculation of sample concentration based on the standard curve.
Since serum contains substances that could potentially interfere with the measurement (I'm thinking of lipids), and since serum is highly concentrated in protein (60-80 mg/mL), and since the dynamic range of the Bradford assay is limited (and depends on the source of the reagent), it will be necessary to dilute the serum samples substantially to stay within the dynamic range of the assay and avoid interference by lipids.
Using a commercial Bradford reagent, in my experience the standard curve might go from 0-10 micrograms of bovine serum albumin per well in a 96-well plate with 10 µL of sample + 200 µL of 1X dye. For a diluted serum sample to have 5 micrograms in 20 microliters (0.25 mg/mL), you would dilute the serum by a factor of 240-320-fold.
Contaminating component that reacts with the "Bradform reaction" similar to the Lowry assay; one would need to have a detailed content listing of your solution conditions. to identify the "culpret".
We abandoned Bradford and its derivatives early after its published announcement.
BTW: our standard curves routinely provide regression analyses R^2 of 0.9985 or better - reproducably over 3.5- to 4-orders of magnitude for protein content - which covers crude homogenates to (column) putified proteins - single bands on silver-stained SDS-PAGE.
Color in the Bradford is due to the dye binding to protein. Interference can come from anything interfering with that process, either by binding to the dye without causing the color shift or by binding to the protein and preventing the dye from binding to it, thus giving a lower absorbance at 595. At 20 ul of serum you are about 10-100X the useful upper limit of the Bradford assay.
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