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