Re: [ccp4bb] very high concentration of protein

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Shredded E coli pellets
From: Filip Van Petegem filip {- dot -} vanpetegem {- at -} GMAIL {- dot -} COM
Date: 2009-07-02

Next message:
Subject: low UV reading on AKTA prime
From: Palm palm {- at -} UNI-GREIFSWALD {- dot -} DE
Date: 2009-07-02

Subject: Re: very high concentration of protein
From: Filip Van Petegem filip {- dot -} vanpetegem {- at -} GMAIL {- dot -} COM
Date: 2009-07-02

Dear Peter,
it's a common phenomenon to create protein concentration gradients inside a
protein concentrator. Simply take it all out of the concentrator, put it in
a separate tube, and mix thoroughly/vortex. The 'slime' may very well
redissolve and you'll a homogeneous distribution. What you describe happens
a lot to us with proteins that don't precipitate and that crystallize
afterwards.

Cheers

Filip Van Petegem

On Thu, Jul 2, 2009 at 8:53 AM, peter hudson
wrote:

> Hello all
>
> I am working with a small protein-protein complex. This complex express
> quite well . I purify in a buffer of pH=9.0 with 150mM NaCl and 1% of
> glycerol and able to concentrate upto 20 mg per ml. I have a two clones of
> this protein complex. One is N-terminal His tagged and another C-terminal
> His-tagged. While concentration of the N-terminal His tagged protein in
> cnetricon it forms yellow color slimy deposition on the surface of membrane.
> while C-terminal His tagged protein does form very highly viscous layere at
> the surface of membrane but it is completly colourless.I aliquate the
> concentrated protein by pippetting into different aliquate rather than
> collection of whole protein by centrifugation. if i check the concentration
> of the last aliqoute(which isbottommost viscous protein part) both prtoein
> complex, it shows very high concentration compared to the first fraction. I
> have not done DLS.
> Is my both C-terminal His tagged tagged as well as N-terminal His tagged
> protein are forming soluble aggregates.
>
> I would appreciate the help.
>
> Thanks in advance
>
> Peter
>

--
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email: filip.vanpetegem@gmail.com
http://crg.ubc.ca/VanPetegem/

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Shredded E coli pellets
From: Filip Van Petegem filip {- dot -} vanpetegem {- at -} GMAIL {- dot -} COM
Date: 2009-07-02

Next message:
Subject: low UV reading on AKTA prime
From: Palm palm {- at -} UNI-GREIFSWALD {- dot -} DE
Date: 2009-07-02