Re: [ccp4bb] Protein-antibody complex

- 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: degradation of protein durring freez thaw
From: Roger Rowlett rrowlett {- at -} COLGATE {- dot -} EDU
Date: 2010-04-22

Next message:
Subject: Re: degradation of protein durring freez thaw
From: Mischa Machius machius {- at -} MED {- dot -} UNC {- dot -} EDU
Date: 2010-04-22

Subject: Re: Protein-antibody complex
From: Guenter Fritz guenter {- dot -} fritz {- at -} UNI-KONSTANZ {- dot -} DE
Date: 2010-04-22

Hi Jan,

you might mutate the Cys residues which are oxidation sensitive,
you could block the Cys thiols in your oxidation sensitive protein,
or you try crystallization at slightly acidic conditions where the Cys
thiol should be more stable (might be bad for the ab-protein complex),
or try crystallization in a glove box.

But before doing all this you might check whether the thiols of your
protein react with the disulfides in the ab. I don't think this happens
frequenetly, but we had one case where a solvent exposed Cys thiol
attacked the disulfide in an Ig domain. This happened only at high
protein concentrations (>10 mg/ml). The not desired result was a
covalently Cys bridged protein-Ig complex. For a test you can
concentrate ab and protein separately, mix them, take aliquots at
several time points, and run non-reducing SDS-PAGE. If you get a new
high MW band you can do tryptic digest-ms analysis to identify the
responsible Cys residue.
Another control experiment: just incubate your protein without DTT,
take aliquots at several time points, and run non-reducing SDS-PAGE.
Again mass-spec should give you an idea which Cys is reactive.
Mutating only this Cys might be already sufficient.

HTH
Guenter

> Hi All,
>
>
>
> I have a simple question about the complex formation between
> macromolecular complex and antibody. My protein is stable in the
> presence of the 5mM DTT and under these conditions the reducing
> environment is too strong for the antibody to survive. I am also now
> trying to check the stability of the protein in lower molar
> concentration of DTT, but as DTT being a strong reducing agent it
> might still pose a threat to the disintegrate the antibody.
>
>
>
> Does anybody have experience in handling protein-antibody complexes
> using other reducing agents? Your answers and help in this regard will
> be highly appreciated.
>
>
>
> Thanks,
>
>
>
> Jan
>

--
***********************************

Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz

Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz

e-mail: guenter.fritz@uni-konstanz.de

Phone Office: +49-(0)7531 88 3205
Phone Lab : +49-(0)7531 88 3733
Fax: +49-(0)7531 88 2966

CCP4bb navigation

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

Previous message:
Subject: Re: degradation of protein durring freez thaw
From: Roger Rowlett rrowlett {- at -} COLGATE {- dot -} EDU
Date: 2010-04-22

Next message:
Subject: Re: degradation of protein durring freez thaw
From: Mischa Machius machius {- at -} MED {- dot -} UNC {- dot -} EDU
Date: 2010-04-22