HIS-tagged protein purification - (Jun/07/2011 )
Hi everyone, It's the first time I've joint the forum. I am now trying to purify my HIS-tagged protien. But I have a problem. After eluting, I did electrophoresis on SDS-PAGE. The result seems very good, there's just a band of interest. But after eluting step, I eliminated immidazole by using the PD-10 desalting column and then did electrophoresis again. It's so bad because many other bands appear. I did every step very carefully of course. There is anyone can tell me how to improve the bad result. Thanks sincerely for your kind help.
#9
The protein is very difficult to purify which is why I decided to go with his tag to begin with. The odd thing is that I attached the his-tag with all the different linkers in between including a long glycine polylinker and it won't bind. Also wouldn't protein be completely unfolded and thus histag exposed when you do SDS PAGE gel before you do Western blot?
thanks for the post. Misery loves company.
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I wasn't getting any binding to the column so I suppose there was not protein when I ran my elution on SDS-PAGE/WB. Any luck until now with that protein?
sashacat on Tue Jul 12 20:17:30 2011 said:
The protein is very difficult to purify which is why I decided to go with his tag to begin with. The odd thing is that I attached the his-tag with all the different linkers in between including a long glycine polylinker and it won't bind. Also wouldn't protein be completely unfolded and thus histag exposed when you do SDS PAGE gel before you do Western blot?
thanks for the post. Misery loves company.
Before further troubleshooting, had you tried using your whole "cloned and expressed E. coli cells" (not the purified protein) and run SDS-PAGE & WB? DO you see your wanted band in this case?