3 replies to this topic

[tbrodeur]

I am purifying a His-tagged toxin fragment whose three tryptophan residues have been replaced by 5-fluoro-tryptophan.

Following the concentration of the column fractions, I dialyze from 100 mM imidazole/500 mM NaCl/20 mM Tris ph 7.9 into 50 mM NaCl/20 mM Tris pH 7.4 overnight.  The protein precipitates out of solution and I have been unable to resuspend it.  I suspect the aggregation is due to hydrophobic interactions, since the wild type tryptophan protein does not precipitate.

I have tried increasing the NaCl concentration to 500 mM but this has not increased solubility.  I would appreciate any suggestions as to what changes in the buffer might prevent protein precipitation.

Thanks!

[scogne]

Hi,
did you check the pI of your protein of interest?? It must be different from 7.5 to use these buffers, if it is not your protein will of course be aggregated...

[Jenny]

Try to dissolve in ureabuffer!!

[Sciad]

Protein precipitates at isoelectric point. Try changing the pH if you can (I assume it is in solution at pH 7.9, i. e. the previous step).