Hello. I used from Thermo Scientific BCA kit and Sigma protein STD 2 mg/ml (0-2000 ug/ml) and prepared a lot of dilutions from my sample (up to 1-64). I had a different final concentration on my samples (I used from Gen 5 software for calculation of my samples concentration *dilution).
my raw data have been attached.
It is because of the range of the standards when you use the BCA assay. With this assay, you set your standards between 0.1 mg/mL and 1.0mg/mL, and only somewhere within this range do you actually get a direct proportion between absorbance at 582nm and protein concentration. Higher than that and you have too much error due to the inner filter effect, and lower than that you have too much error due to electronic noise. It is only when the actual absorbance of your sample dilutions is within the linear range of the absorbance of the standards that you can trust the reported concentration. Note that your reported concentrations are coming out to around 11 mg/mL using your more dilute samples: if that is so, then at 1:80 dilution the actual concentration of protein in the tube is about 0.14mg/mL, which is right there in the range of your standards. Likewise, the 1:40 dilution is about 0.28 mg/mL, which again is in the range of the standards. However, your 1:10 dilution is outside that range already, and the 1:5 dilution is far, far removed. The reported values can therefore not be trusted, except for your greatest dilution factors.
As for the curious disagreement that your first measurement is always reporting a lower concentration than your second, this is an artifact of the BCA test that shows up if you had any significant gap of time between the first and second measurements or between the preparation of the first and second samples. The BCA reagent is highly unstable and continues developing the chromophore without any protein, especially at temperatures above 30 C. If one of your samples was prepared and measured closer in time to when you measured the standards, it is more to be trusted.
Benjamin Lasseter : thank you very much for your complete explanations.
Dear Zahra
as Benjamin told the problem is the range of concentration.
You need to consider in your analysis only samples and BSA standard that supply a Absorbance value in the range 0,1-1.0 because Absorbance upper than 1.0 are outside the instrument linearity.
good luck
Manuele
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