i am trying to purify a C-terminal 6x-his tagged 100 kDa human helicase from BL21(DE3) codonplus strain using Ni-NTA affinity chromatography.
i have followed the published protocol and add bezonase to remove DNA/RNA, add 0.5 M NaCl, 0.2 % triton x-100, 0.5% chaps to remove nonspecific interactions. the yield is good but i get persistent 60 kDa-70kDa bands and i have verified by immunoblot with anti-his antibody that these are not the degraded fragments.
i tried to wash the column with 5 mM Mg-ATP to remove the HSPs but examining the elution fractions i found that instead i have lost my helicase too !! i mean the elution fractions showed no bands even in silver staining.
any suggestions would be helpful and thanks in advance
Hi, Are you sure HSPs are there? according to (Ochs, Protein Contaminants of Sodium Dodecyl Sulfate-Polyacrylamide Gels, Anal. Biochem. 1983, 135:470-474 and Riches et al. Contaminant bands on SDS-polyacrylamide gel electrophoresis
are recognised by antibodies in normal human serum and saliva, JIM,1988) the contamination that you have observed maybe some skin proteins which have been added to your sample during sample loading. the skin contaminants are as large as contamination which you are encountered. I suggest to make a new sample loading buffer and electrophoresis tank buffer before deciding about contaminants.
thanks sebastian and kazem. yes i am using codonplus RIL. these bands are too thick to assume human contamination. i have aliquoted 20 uL of the NiNTA resin and checked on a SDS gel to see if the protein precipitated on the resin, but couldn't find any bands there too
well sebastian, i think it could have washed out with the mg-atp fraction, which i did not save, as i was not expecting it to wash off. i will upload a pic soon. thanks for your interest and help
One trick it to take the Ni-NTA elution fraction containing your His-tagged protein and contaminants and dilute 1:2 to 1:3 (if it does not bind with 1:2 dil., go to 1:3 dil.) in buffer A (buffer w/o imidizole) and then load back onto a clean/new column and elute again with an imidizole gradient. This can dramatically reduce background proteins. This will work if you used a reasonably slow imidazole gradient to elute the His-tagged protein, versus a 500 mM step gradient. The other option is to use a more specific His-tag media like Talon or Complete.
OK, thanks for all your help. i would like to add an update. 2 things helped me for better purification.
(1) i was using qiagen NiNTA earlier. i started getting poor yields when i switched to another brand. this other brand worked excellently for some other proteins and ideally this shouldn't happen but somehow was not effective for this protein. i have checked it twice. moment i switched back to qiagen, i got better yields.
(2) i used the protocol by Rial and Ceccarelli ( http://dx.doi.org/10.1016/S1046-5928(02)00024-4) to remove the contaminating bands.
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