I remove 85 aa from my enzyme(cellulase). I purified enzymes(truncated and wild-type). After dialysis, I run 20 microliter of them for SDS-PAGE. Both of them have the same sharpness on gel, but it's interesting for me that bradford assay is so different. In 595 nm, absorption of truncated enzyme is 0.16, while wild-type is 0.8.
I can't understand difference when the mechanism of binding coomassie blue to aa is the same for bradford and staining of gel.
I think the situation you are describing is that the SDS-PAGE staining indicates that the 2 protein preparations have about the same amount of protein, whereas the Bradford assay indicates that there is a large difference in concentration. However, you mentioned the sharpness of the gel bands rather than the amount of staining, which is the relevant observation. You should use a densitometer to integrate the total amount of stain associated with each band in order to quantify the amount of protein, which is not easy to judge accurately by eye. Ideally, you would load various volumes of each protein sample and plot a curve of stain quantity versus volume to determine the ratio of the concentrations of the two protein samples. A second consideration is that the two proteins have different masses, so each mole of the larger protein binds more stain than each mole of the smaller protein. This has to be taken into account in the calculation. Finally, the amino acid composition can affect the amount of stain that binds.
Regarding the Bradford assay, you should use a standard curve with a protein of known concentration to convert absorbance to protein concentration. Avoid using measurements that are beyond the dynamic range of the standard curve. Again, differences in the amino acid compositions of the proteins could lead to different responses in the Bradford assay.
Hi there,
As Bradford measurment is not reliable from one protein to an other and if your protein samples are pure, I would suggest to perform Nanodrop experiment which is based on specific aa content of each protein. Basically you just need to know the primary sequence of the pritein from which you deduce the molar coefficient extinction at 280nm. So the quantification is specific for every protein considered.
Hope it helps.
Hi,
Also I suggest you used NANODROP experiment as Dominique said. Bradford in several cases can not show the exact amount that you want.
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