Hello folks,
I had a N terminal His-GST-TEV cleavage site tagged protein (15mg) on dialysis over ~40 hours in the presence of TEV protease (1mg) with a buffer change in between. Buffer is 50 mM NaH2PO4, 300mM NaCl, 10mM Imidazole. The dialysis product was loaded on to Ni-NTA beads equilibrated in same buffer (2:1 volume of protein to beads, total volume 10ml in a 25 ml biorad gravity flow column).
I observe a white cloudy precipitate in 30 mins. I'm anxious if I'm losing my tag free protein.. What could this be?!
Cheers,
Pramod
You can centrifuge your sample and run a SDS-PAGE for the pellet and supernatant. If the aggregate is not your protein, you can continue your work. But if your tag-free protein tends to aggregate, you should prepare a different construct (like Sebastian said, maybe try to express and purifiy only His-tagged protein). If you really want to remove the His-tag, you should use pET47b vector. The protein is being expressed with cleavable His-tag. In this case, the HRV-3C protease is needed.
Best regards!
did you tried loading the cell lysate on to Ni-NTA beads?? Instead of doing dialysis load the cell lysate on to the column and wash it with buffer and then do on-column TEV cleavage. Hope this will help!!
I had actually done this before, but with the dialysis buffer and equilibration buffer of Ni-NTA beads post cleavage being 50mM HEPES pH 7.4. It was much better then, and this problem occurred only when I changed buffer to 50mM NaH2PO4 pH 8.0.. I seem to think it could be the buffer (?)
Well, nothing is clear. What is the amount of salt you use? and what is the PI?
Sorry it is 300mM NaCL when i use NaH2PO4 and 150mM KCl with HEPES. the pI of the final protein after cleavage from the His-GST tag is 5.
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