What happens if you load your elution fraction into a size exclusion column? If your protein of interest comes in the void volume together with most of its contaminants, you'd better test a different construct, and that's much more than only changing the tag from N- to C-terminus. Sumo and GST might be solubilizing things that shouldn't really be soluble...
Carlos Em 06/08/2011, às 16:43, Paul Kraft escreveu: > Hi Lisa, > Have you compared your yield of purified protein of soluble protein per gram > of pellet, 4M Guanidine solubilized pellet (wash and elute from Ni column > with 8M urea .5M imidazole..otherwise the gel with run crappy), and and total > protein in the pellet on a page gel? The main reason I would think you get > high background of cellular proteins on you Ni purification is because your > yield is too low (I know obvious..). There are a variety of methods to > increase yield like expressing at RT (assuming your expressing in E.coli), or > switching to yeast, insect, or mamallian cells if your protein is of human > origin. The most helpful and easiest method to try first (after trying low > temp), would be switching the tag from N to C terminus or vice versa. > Otherwise switch to a thermophile version of your protein if possible (and > try cysteine mutants or other bacterial organisms for source the source gene). > Paul > ps one thing I forgot, you mention it is a DNA binding protein, solublizing > in 2M NaCl is much better than 1M NaCl, you could just be losing it...the > protein that is :-) > > Dr. Paul Kraft > Structural Biologist > cell 586-596-2770 > email: [email protected] > email: [email protected] > > > > > This communication and any attachments contain information which is > confidential and may also be privileged. It is for the exclusive use of the > intended recipient(s). If you are not the intended recipient(s) please note > that any form of disclosure, distribution, copying or use of this > communication or the information in it or in any attachments is strictly > prohibited and may be unlawful. If you have received this communication in > error, please notify the sender and delete the email and destroy any copies > of it. > > > E-mail communications cannot be guaranteed to be secure or error free, as > information could be intercepted, corrupted, amended, lost, destroyed, arrive > late or incomplete, or contain viruses. We do not accept liability for any > such matters or their consequences. Anyone who communicates with us by e-mail > is taken to accept the risks in doing so. > From: LISA <[email protected]> > To: [email protected] > Sent: Thursday, August 4, 2011 11:51 AM > Subject: [ccp4bb] gst-tag protein purify problem > > hi guys, > I have a DNA binding protein and expressed the DNA binding domain (150 aa) > with his-sumo tag or gst tag at the n-terminal. I tried to purified it with > Ni column or Gst column separately. But purity is lower than 50% after Ni or > GST column. This protein only stable with 1M Nacl or higher. I worked on it > almost half year. But I still can not get the pure protein. > please give me some suggestions. Thank you. > > Lisa > >
