Hello
I have purified my protein by Ni-NTA column chromatography. i've used gradient elution in elution step. after collecting the fractions i should read the bradford absorption of fractions to find out the amount of eluted protein.
But i don't know the imidazole concentration in each fracion. so my question is:
How should i read the bradford absorption of elution fractions from Ni-NTA column chromatography while i dont know the imidazole concentration exactly? what is the blank for each fraction?what is the imidazole concentration for each fraction?
Thanks in advance.
Imidazole should be compatible with the Bradford assay; most companies state an upper limit of 200 mM. Generally a his-tagged protein will elute at a concentration below that (70-100 mM).
One way to be sure though would be to run some blanks with at least three different concentrations within the range of your concentration gradient---say 0 mM, a middle value, and the maximum mM value---and you should get a relatively linear increase in absorbance at 590 nm if it does interfere. Assuming you used a linear gradient, you should be able to determine the contribution from imidazole interference to the total absorbance of the dye complex.
The concentration of Imidazole is an issue with BCA, with Bradford you don't need to worry about the concentration of Imidazole. Bradford is more tolerant to high salt concentration hence working solutions of Bradford can be used with eluted fractions directly to measure the concentration of proteins.
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