protein precipitation - (Sep/07/2006 )
I am working with a recombinant his tagged reductase protein. This protein remains stable in the elution buffer for 3-5 hours but after that it precipitates out. This is there when even kept at 4oC. Freezing enhances the precipitation and it immediately ppts at -20 and -70oC. It remains stable at these temperatures when kept in 40-50% glycerol for more than 2 months and the activity is also retained.
I am trying to crystallise this protein for which i need to remove glycerol. I have dialysed it against 20mM tris+ 100mM NaCl+2mM DTT but the protein does remain stable for 1-11/2 day thereafter it precipitates. This protein has got 5 cystein residues. Please tell me if there is any way out?
I would try to reduce ionic strenght in dialyses buffer to reduce hydrophic interactions which might force precipitation; what is known about pI? one should also have a sharp look on buffer pH; half or fourth of DTT ought also do
I wonder if adding low amt of surfactant would help. 0.01% Tw80, for example.
thanks for replying. i have tried low ionic strenghts even as low as 10mM tris, 20mM NaCl . as far as DTT is concerned as i had mentioned earlier i have tried 2mM DTT. May be i will try to increase it. The theoretical PI of me protein is 6.8 (this value is without the addition of His tags). I have been trying buffers with PH 8.0.can you suggest something else.
thanks for replying
i only know that the addition of surfactants would prevent the precipitatation but i don't know how actually it works? work the surfactant cause denaturation of protein. i want protein in native state.