I have lysed my e. coli cells (after adding protease inhibitors to the lysis buffer) and freezed the lysate. I then unfroze with the intent of purifying my protein , however due to column technical errors I ended up storing it O.N at 4°C with the hope that the next day after washing the column it would work again. I was worried that unthawing would be detrimental.
Long story short, the lysate has been in the fridge for 3 nights. It is still clear, however there is a faint pellet at the bottom of the falcon.
Is there any hope this protein has survived? Should I go ahead and try to purify this lysate or should I throw it away?
It all depends on how stable is your protein and how valuable it is. For example, if the protein is unlabelled and easy to produce I would start with another growth, otherwise there are 2 option: you can put it on an SDS page and check if there is still a clear over-expression band, or run the first step of purification and then run SDS page. Growing the cells requires an extra day, if I was you I would try and purify it while setting up a new growth.
Thanks for your reply Domenico. It has a His-tag and it is quite stable I suppose.
I think the best action in your situation depends also on other factors, including your intended purification scheme, if the protein's folded structure is critical, and downstream applications. Running the suggested SDS-PAGE is a good first step, regardless. If this you're doing any sort of structure determination or enzymatics, I'd scrap this batch and start over. If you're using the protein for something else, it may still be worthwhile to move forward with, but that depends on how pure you need it and the purification scheme you'll use. For example, if you're using affinity chromatography as your only separation method, you'll also capture and co-purify partially-digested fragments of your target protein that still bear the His-tag.
Long story short, I'd mostly likely choose to start over from scratch unless it's either super tedious/expensive - but this, again, depends on how the protein will be used in the end.
Hi Ross. I am using it for crystallography so I think it is probably best to start over. Or, purify it at some point just out of curiousity to see what happened.
Thanks!
Hi there,
As there is absolutely no golden rule concerning protein stability in a lysate context, I would suggest you to have a go with the purification and see what the protein looks like at the end of it. It would still be possible to redo everything from the start once you know the result...
Hi again, Engi - sorry for the delayed reply, but it seems you're already well on your way. For crystallography, I think you've certainly made the right move in starting fresh. Best of luck!
Update for all: Storing the lysate at 4°C was fine, as I had the same yield of protein as I had when freezing the lysate.
Which was not much anyways, but point being it did not affect the yield and it crystallized at the end..
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