I am purifying a viral protease of size 33 kda in E.coli BL-21 expression system. After cell lysis due to some unforeseen technical problems I was unable to purify my protein of interest on the same day. Can I continue the His-tag purification protocol on Ni-NTA column the next day with the same sample ? How can I store the now lysed, not-purified sample so the the protein loss is minimum ?
Try to freeze the sample at with liquid nitrogen and then keep it at -20 over night. If you are no able to isolate the same day it is better to centrifuge the bacteria to a pellet and freeze it.
Yes, I would definitely go with Sebastian's tip to freeze the bacterial pellet.
It seems to me from the wording of your question, however, that you have already lysed the sample? The stability of your expressed protein (probably unknown) is going to be critical here. I would definitely snap freeze the lysate in liquid nitrogen a.s.a.p., and store at -70, but it may be too late to save the prep. You could simply start again, or take a chance with the purification, with the risk that you may have some degradation.
Thank you Nick and Sebastian. I will obviously freeze the pellet as I do any other days. In this case however, I will take a chance at the purification before starting the whole process from the begining.
Freeze everything using liquid N2 or He, and defrost whenever you want to resume your work.
I usually add PMSF and TCEP when lysing samples to inhibit protein degradation and prevent the protein from oxidation. Then the lysed samples are immediately frozen in liquid nitrogen and stored at -70 ℃. Thaw samples slowly when thawing and avoid repeated freezing and thawing. If you are purifying the target protein for the first time, it is recommended to purify it immediately after lysing the cells.
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