I have been struggling to determine the protein concentration, which is crucial for optimizing the working concentration needed for my research assays. Unfortunately, I have been receiving conflicting advice from those around me, which has added to my confusion. I urgently need a clear and reliable method to calculate the protein concentration so that I can proceed to the next phase of my research.
Dear Guidani
if you subtract the aborbances of the samples for the one of the blank (bradford plus buffer) the trendline have to intercept zero more or less. However, since the bradford has intrinsic assorbance you will not intercept the zero in case you do not substract the sample absorbance for the blank one.
best
Manuele
You can still use your standard curve if it doesn't go to zero. The only thing that matters is that you can draw a line (or curve as long as you know how to solve the curve equation), using standards with known concentrations. Then place the absorbance of your unknown sample somewhere on the line to visualize its concentration. And of course the sample should be measured in the same experiment in parallel with the standards.
The Bradford assay has been confusing grad students for generations. I should warn you that the Coommassie stain reacts differently to different proteins, so it may not agree with more accurate techniques such as A280 measurements. However, it can be useful when A280 isn't an option.
Jonathan Wagner thanks you for your input and ill be more aware of the warning you posed too
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