Re: [ccp4bb] precipitation of the protein in crystallisation solution

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

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

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Subject: ccp4i problem - bad marshal data
From: Jan Abendroth jan {- dot -} abendroth {- at -} GMAIL {- dot -} COM
Date: 2009-06-02

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Subject: Re: deglycosylation of proteins expressed in mammalian cells at room temperature or lower
From: Pius Padayatti ppadayatti {- at -} GMAIL {- dot -} COM
Date: 2009-06-02

Subject: Re: precipitation of the protein in crystallisation solution
From: Matthew Alan Bratkowski mab259 {- at -} CORNELL {- dot -} EDU
Date: 2009-06-02

Hi Peter,

You first want to reduce the protein concentration to avoid rapid
precipitation upon adding crystallization solutions. Try concentrations
of 5 - 10 mg/mL instead of 15 mg/mL. Next, consider changing the NaCl
concentration. Salt can act as a co-precipitant, and lowering its
concentration may increase solubility. Conversely, some proteins
precipitate badly when salt is dialyzed out, so increasing the salt may
also help. You also may consider using a different pH or different buffer
(HEPES instead of Tris for example).

Glycerol is supposed to make proteins more soluble, but proteins that I
have worked with have turned yellow when concentrated in the presence of
10% glycerol. Try dialyzing out the glycerol or using a buffer that does
not contain any glycerol and see if you still see a yellow deposit on the
concentrator or not. I think that the yellow color is caused by metal
impurities in the glycerol, so using a higher purity glycerol may help
too.

If none of these suggestions help, you may want to consider using a fusion
tag (maltose binding or SUMO) tag for one of your proteins. These tags
can help increase protein solubility, but unfortunately may also disrupt
protein-protein interactions.

Matt

CCP4bb navigation

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

Previous message:
Subject: ccp4i problem - bad marshal data
From: Jan Abendroth jan {- dot -} abendroth {- at -} GMAIL {- dot -} COM
Date: 2009-06-02

Next message:
Subject: Re: deglycosylation of proteins expressed in mammalian cells at room temperature or lower
From: Pius Padayatti ppadayatti {- at -} GMAIL {- dot -} COM
Date: 2009-06-02