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 <[log in to unmask]> (on behalf of Paul Kraft <[log in to unmask]>)
>Subject: Re: [ccp4bb] gst-tag protein purify problem
>To: [log in to unmask]
>
> 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: [log in to unmask]
> email: [log in to unmask]
>
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> --- On Sat, 8/6/11, Carlos Kikuti <[log in to unmask]>
> wrote:
>
> From: Carlos Kikuti <[log in to unmask]>
> Subject: Re: [ccp4bb] gst-tag protein purify
> problem
> To: [log in to unmask]
> 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, ās 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: [log in to unmask]
> email: [log in to unmask]
>
> 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 <[log in to unmask]>
> To: [log in to unmask]
> 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
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