I'm diluting 1, 2, 4, 8, 16, 32 & 64 ul of 10mg/ml of BSA solution in to 99, 98, 96, 92, 84, 68 & 36 ul of PBS buffer, making total 100 ul. Now adding 50 ul of Bradford reagent. I'm wondering whether BSA concentration should be calculated from 100 ul or 150 ul.
Hi there,
For simplicity, standard curve should always be OD=f(µg of BSA). If doing so your question would be pointless as any meaured OD would correspond to one single amount of protein. Knowing what volume of the protein sample has been introduced in the assay, one simple division will give you the actual concentration of the sample.
Hi.
As Dominique pointed out exactly, do not worry. In most cases of Bradford assay, it would be better to add large vol of higher stock protein dye (i.e., 1,500ul) to smaller vol of standard or unknown target protein sol (i.e., upto 50ul), eliminating or minimizing possible dilution effect(s) of Bradford reagent in the mixed solution form next time. Do forget blank.
Regards.
The standard curve should be based on the number of micrograms of protein, as Dominique Liger said, rather than the concentration of the standards. This reduced confusion.
The volumes of sample (or standard) and Bradford reagent must be identical for every sample. Also, the composition of the sample and standard should be the same, if possible. If not, then it should at least be ascertained that nothing in the sample that is not also in the standard interferes with the assay.
Your method differs from the one I use. I agree with Sang Ho Lee that the sample or standard volume should be small compared to the volume of Bradford reagent (unless you are using concentrated Bradford reagant). In my case, I use a 10 µl sample and add 200 µl of 1X Bradford reagent. My standard curve is from 0 to 5.5 µg of BSA and is linear. The standard curve can be extended to larger amounts of BSA, up to 11 µg, but then becomes nonlinear and is fit nicely by nonlinear regression to a second-order polynomial.
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