Other than using a positive control, is there a way to determine if the Genscript anti-his elisa protein quantification kit underestimated the protein concentration? Because the discrepancy using the absorbance of 280 nm method + running on gel greatly differs from the protein quantification using Genscript?
If anyone uses it, can you show me the calculations you do? (eg, taking into account molecular weight of the standards in the kit against your own peptides)
Protein quant methods often vary in standard curves by up to 2-fold, sometimes much more. For example, OD280 is based on Trp content, and gel scanning is based on the amino acids that work for whatever stain you use.
You generally have two choices. One is to get enough protein that you can desalt it, lyophilize it, and actually weigh it on a balance. Then create standard curves using your assay with dilutions of known mass in a standard solvent/buffer. However it is often more practical to create your standard curve using a more available purified protein and then report the method used.
If you are measuring the concentration of a purified protein, then you can try using other common protein assays, such as Lowry, Bradford and BCA. Of course, for these you must choose a protein to use as the standard. Bovine serum albumin is often used, but if your protein has an unusual amino acid composition, you may want to try using a different standard that is a better match, such as IgG.
If you are trying to measure the concentration of the His-tagged protein in a mixture with other proteins, then the above approach will not work unless you know the percentage of the protein that is the one of interest (i.e. the purity). Estimating the purity of a protein by SDS-PAGE by eye is very unreliable, but it can be done with a reasonable degree of accuracy by using a densitometer, as long as the protein of interest is a major band.
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