Good point. And solubilizing tags can sometimes drag unfolded garbage into solution with them.
===================================== Phoebe A. Rice Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp ---- Original message ---- >Date: Mon, 8 Aug 2011 15:26:43 -0700 >From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> (on behalf of Paul Kraft ><haresea...@yahoo.com>) >Subject: Re: [ccp4bb] gst-tag protein purify problem >To: CCP4BB@JISCMAIL.AC.UK > > Forgot to add, a 150aa dna binding protein should > only need a minimal hexahistidine tag....these > proteins are typically very soluble and express > well. Adding a GST or sumo tag is overkill. > > Dr. Paul Kraft > Structural Biologist > cell 586-596-2770 > email: haresea...@yahoo.com > email: kraft_proteome_resea...@yahoo.com > > 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. > > --- On Sat, 8/6/11, Carlos Kikuti <kik...@gmail.com> > wrote: > > From: Carlos Kikuti <kik...@gmail.com> > Subject: Re: [ccp4bb] gst-tag protein purify > problem > To: CCP4BB@JISCMAIL.AC.UK > Date: Saturday, August 6, 2011, 5:13 PM > > 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: haresea...@yahoo.com > email: kraft_proteome_resea...@yahoo.com > > 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 <science...@gmail.com> > To: CCP4BB@JISCMAIL.AC.UK > 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
