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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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aggregation/monomer-dimer equilibrium SOS
Posted by: pinnochio (IP Hidden, New member, 2)
Date: May 24, 2005 03:47PM
Hi
I have a 12.7 KDa bacterial protein expressed as a C terminal GST fusion in E.Coli BL21 cells. This protein is an excellent expresser but crashes out if it is too concentrated. I use upto 1mM TCEP in my purification buffers and have tried adding 1mM DTT too. But I can still see a dimer band in the SDS-PAGE. This would not be so much of a problem since I'm doing gel filtration and colelcting only the monomer peaks. But MALLS data of the protein indicates that it exchanges between a monomeric and dimeric form at an intermediate rate. This is a problem because my goal is to determine the structure of this protein. Can anyone please tell me; 1. Ways to avoid the dimer formation in the first place. 2. How to push this monomer-dimer equilibrium that is going on with my protein in one direction or the other so I can determine the structure of either the monomer or the dimer. (preferably the monomeric form) Any other ideas? Thanks, Pinnochio.
Re: aggregation/monomer-dimer equilibrium SOS
Posted by: 5'-ATCG (IP Hidden, Unregistered user, )
Date: May 24, 2005 06:54PM
According to what you're saying it's very difficult to push the reverse way (dimer to monomer) since this is against nature (e.g. concentration-dependemt). Apparently the monomer is unstable so (1) try to put your protein in highly denatured condition (8m urea); (2) or to stabilize it by incorporation of co-factor like ATP, cation ion, DNA/RNA, small peptide or ligand depends on what your protein is; (3) try to shorten your protein....
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