I'm using a phosphate buffer (pH8) as recommended by the protocol of Quiagen (50 mM NaH2PO4 + 300 mM NaCl + 5-10mMImidazol) to get protein purification with his tag from an insect cell (using a Ni-NTA resin and batch method). I added detergent NP-40 1% or Triton X-100 1% to lysed the pellet. Is triton better at lower percentage, like 0,1%? When I did the purification with 1% triton, the Bradford concentration was wrong and I had triton on my elution protein. I have had close to 4-5 mg of protein from 1,8 L (3 million cell/ml). Is this quantity good? I do not know if it is necessary for re-purification after this, maybe I can obtain a bit more of protein. Is possible that triton is interfering with my purification?
I have a question regarding two more steps which I do routinely. I always pick up the pellet at 5000g and 10 minutes and then freeze the pellet without any protective buffer. Is this wrong?After lyseing the pellet, I did a one cycle of freeze-thawing and centrifuged at 10000g for 15 minutes and there is always a white mucus that I think could be DNA (descarded). Is it DNA?
Please advise.
Hi Roman,
That is a lot of questions. I have expressed and purified a few different proteins from Insect cells. Easiest questions first.
Yield - from the proteins I have purified anything around 4mg/L is great. The one that I have purified for years was generally around 1-4mg/L as it depended on how well the transfection went and when you harvested.
Protection buffer - I am unsure if this is needed. I have used PBS and no PBS and found no difference. It could be insect cell line dependent as I have mostly used Sf9 cells.
Lysis and Triton - I prefer syringe and detergent lysis as I am not a huge fan of freeze/thaw. I just dont believe that one cycle of freeze thaw is enough to break all the cells open. Combining both I found gives me a good yield. I use exactly the same buffer as you with 1% TX100 and 10% Glycerol. I only use TX100 (0.1%-1% gave me minimal difference) in the lysis buffer so by the end of the purification (after 100mL wash with 10mM Imidazole and 50mL of 20mM Imidazole) there should be minimal detergent left. I have not tried to test at lysis stage however. You would have to dilute the lysis so much to make an accurate Bradford result (too much protein) that I would be surprised if it interfered. Also I am not a huge fan of Bradford. Personally I prefer A280 but every method has its pros and cons and I think the more important thing is consistency.
DNA - the easiest way of determining if it is DNA is to add Dnase to the lysis buffer. (I routinely add Dnase anyway so do not often see what you are describing.) If it goes away then I would suggest that it is DNA.
Purity - Not sure what you mean but I would run it onto a gel and see what the purity is like. Then generally follow up with a FPLC step if it is still impure.
Hope this helps,
Russell
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