problems with loss of purified protein due to precipitation - protein purification (Sep/12/2004 )
I am trying to purify a ~30 kDa 6xHis-tagged protein via Ni-NTA columns and am having big problems with protein precipitation.
I am purifying under denaturing conditions (8 M urea), and subsequent to the Ni-NTA column am desalting with PD-10 desalting columns (Amersham). Following the desalting step, my protein falls out of solution and thus gets lost - almost completely.
I am eluting from the PD-10 column with a Hepes-buffered salt solution (pH 7.4), containing BSA and glucose to help prevent protein aggregation.
Does anybody know of a better way to desalt, or would have suggestions how to prevent protein precipitation?
Would it for example help to increase the volume of the final elution buffer (does anybody know of bigger desalting columns)?
What else could I add to the buffer, with paying attention to the fact that I will be applying the purified protein directly to cell culture afterwards?
Just a stab, but if you are going from 8M urea to none so quickly could your protein be aggregating into insoluble precipitates? If the protein is denatured then suddenly has no urea around to stabilize the hydrophobic regions the proteins will certainly miss fold and will probably misfiled with each other thus forming aggregates.
I could be wrong as I've never used PD-10 columns like that, ut if that is the problem you could try dialysis and even stepped gradual dialysis: first against say 6M urea, then 4 then 2 etc until you get rid of all of the urea.
Hope that helps
If you can tell me about the solvents you are using, probably I can help.
Thanks for your response. I think that this is the most probable explanation and will try dialysis with "Slide-A-Lyzer" cassettes from Pierce next.
Still, PD-10 columns would be more convenient to use, since faster, and it would be great to find a way to prevent protein aggregation also in this method. I tried adding 20% glycerol and 2 mM glutathione, will see what this gives...
Also, the degree of precipitation seems to vary from prep to prep, it is somehow hard to determine how concentrated one can purify the protein without loosing it.
Just saw the response in which you are asking for the solvents:
Up until now my elution buffer for the PD-10 column contained (in mM) 110 NaCl, 4.7 KCl, 1.2 KH2PO4, 20 Hepes, pH 7.4 with the addition of 0.1% BSA and 15 mM glucose to stabilize the protein.
Last time, I added 20% glycerol as a cosolvent and 2 mM glutathione to help correct refolding.