Re: [ccp4bb] very high concentration of protein

- Protein crystallography

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

Previous message:
Subject: Re: multi-domain protein with identical tertiary structure
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2009-07-02

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From: Jacob Keller j-keller2 {- at -} MD {- dot -} NORTHWESTERN {- dot -} EDU
Date: 2009-07-02

Subject: Re: very high concentration of protein
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2009-07-02

Peter,

If I understand what you are saying - then it is very likely that your
protein forms aggregates. Whether this happens on concentration or not is
unknown because concentration may simply bring the pre-existing aggregates
to the membrane.

You can try to make concentration process 'easy' on the protein by avoiding
local over-concentration (formation of a gradient in the centrifugal
device). This can be done by using the inverted (upward-concentraring)
concentrators (such as the Millipore variety - they don't eliminate the
gradient completely but are nevertheless a step above the
downward-concentrating versions) or even better by using a stirred cell etc.

It is generally a good idea to test your protein's oligomeric state and
homogeneity before and after concentration using e.g. analytical size
exclusion or light scattering (or analytical centrifugation, or whatever
else you may have in the lab).

Artem

"Nothing is built on stone; all is built on sand, but we must build as if
the sand were stone"

Jorge Luis Borges

_____

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of peter
hudson
Sent: Thursday, July 02, 2009 10:53 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] very high concentration of protein

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

CCP4bb navigation

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

Previous message:
Subject: Re: multi-domain protein with identical tertiary structure
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2009-07-02

Next message:
Subject: Shredded E coli pellets
From: Jacob Keller j-keller2 {- at -} MD {- dot -} NORTHWESTERN {- dot -} EDU
Date: 2009-07-02