Hello Qiangmin,
Many membrane proteins like to aggregate in detergent, which
will prevent efficient site specific proteolysis. If you have not
already done so, I suggest running a size exclusion column first to
verify that your protein is monodisperse in your detergent and buffer
of choice.
I have also heard from a few other researchers that GFP fusion
can sometimes promote aggregation. I'd appreciate hearing from others
how often they have encountered GFP-mediated aggregation, whether with
membrane or soluble proteins.
Ho
Ho Leung Ng
University of Hawaii at Manoa
Assistant Professor, Department of Chemistry
[email protected]
On Mon, Apr 8, 2013 at 1:01 PM, CCP4BB automatic digest system
<[email protected]> wrote:
> Date: Tue, 9 Apr 2013 01:22:36 +0800
> From: Qiangmin Zhang <[email protected]>
> Subject: Anyone has experience with digesting membrane protein by precession
> protease
>
> Hello everybody,
> I just purified a membrane protein tagged with GFP, which has a cleavage site
> of precession protease. And I got a problem with removing the GFP tag by
> precession protease (1mM DTT and 1 mM EDTA were included in the buffer). It
> can not cut my protein. I have already tried to digest it in different
> detergent like DDM and C12E8 (also different concentration for detergent like
> lowering the detergent to 1.1 x cmc since a recent science paper lowered the
> detergent to this level and got it worked). I know this depends on the
> different proteins. I am wondering if anyone has this experience in digesting
> membrane protein by precession protease. Any suggestions are appreciated.
> Otherwise I might have to go back to just his-tag if there is no trick for
> that. Thank you so much in advance.
> All the best
> Qiangmin Zhang
