Hi everybody,
I'm facing a problem with the purification of an His-tagged protein produced in Sf9 cells. Protein, which is also GFP-tagged, is nicely expressed (unlikely cytosolic), easily extracted from the cells (I used 1% TX-100), perfectly bound the Cobalt column but is impossible to elute... I tried 300 mM imidazole, then 500 mM in the presence of 1% TX-100 and 1 mM beta-mercaptoethanol and finally 1 M imidazole. None of those buffers allowed me to elute anything. I also tried pH5 elution zero again.
If any of you get an idea I'll appreciate because I never faced such a problem before.
Cheers,
:-) guillaume your protein probably precipitates on the column...300mM imidazole is already a lot...
if you put 5mM EDTA over the column, the column will turn white (the c02+ has been eluted and the likely bound prot!) and the eluate will be pink...if the prot is not in this eluate...it means the protein is truly precipitates..so if the protein is soluble in the extraction buffer (when i mean soluble...it must stay in the SN after 45min a 45000rpm) , keep this buffer along your purification including the elution...and think about how to get rid of the triton (i bet this is what you want to get rid ) after the elution...on peut en discuter
Dear Guillaume,
two things that spontaneously came to my mind are ...
1) I agree with Didier about the possibility of precipitation.
2) If it is not precipitation but too strong binding, you could try to shorten the His-tag perhaps from six to four His residues? I would be strongly interested to know if this approach works.
Good luck!
Dear Daniel,
I doubt that strong binding could be involved. Indeed, 6His-GFP is perfectly eluted at 150 mM imidazole (obtained 2 mg from 250 ml infected Sf9). I'm clarifying my lysates at 60 000 g right now to see if my protein precipitates within those conditions. If not I will use same buffer all over the purification process and if it doesn't change anything then will try to strip the column with EDTA.
Thanks anyway and if any of you get other ideas you're welcome to share them.
Cheers.
is sf9 expression absolutly required (any ptm involved!)..if not, parallel cloning and ecoli may give some results.see
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