Reduced temperatures (-20, even -80) do not stop biological processes, they are merely slowed down. So anything that can happen at higher temps will happen eventually at lower temps.

I've worked with one particular protein that tends to aggregate and precipitate during purification. I have noticed that using biomass from frozen pellets that have been stored for a while at -20 leads to an increased amount of insoluble material in my initial extract and lower overall yield.

For other proteins, that behave just fine during purification, I have used frozen pellets that were over a year old with no noticeable loss of yield.

I usually store pellets at -20. -80 after flash freezing might be better, but isn't as convenient! My lab uses HarvestLine Liners from Beckman inside our centrifuge bottles, which make pellet storing very easy - simply decant the supernatant; remove the liner with the pellet in it; heat seal; and stick it in the freezer. To thaw, you just cut the liner and drop the pellet into a beaker with resuspension buffer.

I would echo what many have said already though - don't resuspend before freezing. The main problem I have found is that the cells are now in a specific buffer and if you are trying to optimize purification, might need to spin them back down and change buffer after thawing, but some lysis will have started in the freeze/thaw process.

https://www.beckmancoulter.com/wsrportal/bibliography?docname=DS-8898A.pdf

No limit.. Store at -20 degree..... But its always better to use short storage one.

I imagine stability will vary on a case-by-case basis - so my answer is "Try it and see".

:-D

If your protein is fine, then you know that you can do it next time. If it is not, then you have to make a new batch. I would just be extra careful with the QC of this prep - maybe get some intact mass analysis of the purified protein done.

As a rule of thumb, at -80 It is stable, but in practice it totally depends on the stability of your protein. In this case, the pellet has been stored at -20, thereby I wouldn't be confident using it and I would go for a new culture. As others said, next time quick freeze it in LN and store it at -80!

Nearly no storage limit, even at "only" -20 degree.

Pre-freezing with liqid nitrogen is not necessary

Probably depends on the stability of your specific protein of interest and its sensitivity towards proteases present in the your bacterial cells.

Have you used a cryoprotective agent? If yes , they should last up to 6 months at -20 Cand a bout 1 to 1.5 year at -80 C, if not about a month in -20 would be your best shot, check on Demain and Davies Manual of Bacteriology and Biotechnology to be sure , but just to be on the safe side I would re express them .

As said before, try and see! You can just check with a SDS PAGE if your protein is still there. Frankly, I am very curious to know the result!

The question of the structure and/or the activity of this protein is an other problem. To be sincere, if I were you, I would start another culture just to be sure of the quality of the protein.

So you redissolved the cells and then freeze?

Is your protein an enzyme or do you need to see its activity? if the answer is yes then I probably would not use anything that's been stored in -20c for more than a week. If you only want the protein for something else you can probably use it after a month, but you'd never know unless you have tried it. Rule of thumb is that you should never store protein work that you care about in -80c, and if I were you I'd start from scratch and do the induction again.

Hi! Next time, I would not resuspend the cell pellet in buffer and instead keep it as a solid pellet of cells and it should be stored at -80˚C instead of -20˚C. If that were the case, the pellets should last for 6 - 10 months. Again, this will depend on the protein.

Always go for fast freezing in liquid nitrogen and store at -80C. And keep as a pellet, do not resuspend in buffer - for longer storage. You might resuspend it in buffer but also add some protease inhibitors - for shorter storage

It surely depends on the stability of your protein. You might be lucky and your protein could be fine even after six months at -20°C. Anyway, as others have pointed out, it seems you took the most complicate and probably unsafe route to store your cells. In principle, I would never resuspend them in buffer before freezing and I would not trust keeping them at -20°C for more than perhaps a few of days. Consider that at -20°C the water inside your cells is not completely frozen, and some degradative processes can occur at that temperature. My suggestion would be to freeze at -80° the cell pellet as fast as you can (using liquid nitrogen, if available). At -80° cell pellets are stable for months and possibly years.

Reduced temperatures (-20, even -80) do not stop biological processes, they are merely slowed down. So anything that can happen at higher temps will happen eventually at lower temps.

I've worked with one particular protein that tends to aggregate and precipitate during purification. I have noticed that using biomass from frozen pellets that have been stored for a while at -20 leads to an increased amount of insoluble material in my initial extract and lower overall yield.

For other proteins, that behave just fine during purification, I have used frozen pellets that were over a year old with no noticeable loss of yield.

I usually store pellets at -20. -80 after flash freezing might be better, but isn't as convenient! My lab uses HarvestLine Liners from Beckman inside our centrifuge bottles, which make pellet storing very easy - simply decant the supernatant; remove the liner with the pellet in it; heat seal; and stick it in the freezer. To thaw, you just cut the liner and drop the pellet into a beaker with resuspension buffer.

I would echo what many have said already though - don't resuspend before freezing. The main problem I have found is that the cells are now in a specific buffer and if you are trying to optimize purification, might need to spin them back down and change buffer after thawing, but some lysis will have started in the freeze/thaw process.

https://www.beckmancoulter.com/wsrportal/bibliography?docname=DS-8898A.pdf

Storing your bacterial cell pellet and not as a cell resuspension at -80C is the best way. I have 10 year old cell pellets that we still use to purify protein. Definitely depends on your protein. You won't know for sure unless you try purifying your protein. Storing at -20C is not advisable. In disagreement with the above comment, the best place for your purified protein is at -80C. We use 10% - 50% glycerol to help stabilize the protein. The key is to snap freeze in liquid nitrogen to prevent ice formation.

Freezing and thawing cause lysis to bacterial cells and so they release the protease.

Any way just scrap few cells from your pellets and do a small scale pull down to see whether your protein is still there and start the batch for reexpression as you may be needing more protein .Cells lysed during freezing and thawing hardly give any proteins due to proteases secreted by them even in presence of p;rotease inhibitor as all of protease inhibitors has its own life, 2nd you must check for any fungal growth on cells if it is there then throw the cells.

My experience with a touchy, large ultrapure protein and its ability to onload and offload substrate (highly conformation-dependent) was unequivocal: 4 degrees, a couple of days of decent activity; -20, a couple of months; -80, a couple of years (no end point, project terminated). Go with minus 80 and track samples over time as we did. I was amazed at the fast deterioration at -20, and it was a freezer we took care wasn’t constantly opened and losing temperature stability.

One thing, be sure your storage conditions don’t promote dehydration; keep the samples sealed in glass containers, as most plastics breathe and permit both water extraction and oxidation. And of course, don’t leave the samples sitting around in the ice bucket for a long time before processing.

These are procedures regarding temperature only—proper storage buffer is a whole other subject. I gather you know your target survives both -20 and -80, so developing a standard protocol might include the more stable (-80) temp as you work on some of the other suggestions. Different additives can be a friend to some, and toxic to others. So many proteins, so little time.

I, think your question is not clear. you are not mentioned that your protein is in soluble form or insoluble(inclusion body) form. I am suggesting that once you have to check that your protein stability. If your protein is stable you can carry your experiments.

I am regularly working on soluble and inclusion body proteins only insoluble(Inclusion body) proteins are stable and i am doing my purification work. it is almost stable more than a year, and it is also depends on the protein nature.

But generally soluble proteins are not stable that much longer time when compare with insoluble proteins.

I agree with all the above comments, and particularly with Sourabh Jindal: lysis of bacterial cells upon freezing and thawing may release several host proteins, including proteases. Obviously, this depends on the bacterial type; I assume that you are speaking about some Escherichia coli strain typically used for heterologous expression. Anyway, gram+ (e.g. bacilli) are almost as tricky, in my experience: after a few days storage at -20°C, they are more easily ruptured and even in mild resuspending media they release several of their own proteins and enzymes.

Thanks a lot for all your comments!

I purified protein from 2 pellets - one stored for 6 months and another for 10 months (which was a more unstable protein and the one I was worried about). Thankfully and quite luckily (after reading all the above comments) I got a good yield and the proteins seem to be fine at least on the gel. Of course I still need to see if they are active.

Next time i'll take care to store the cell pellets without resuspension, at -80C.

Cheers!

What if you freeze them overnight and make the protein extraction the next day?

It's 9:00 PM, 50 minutes more to complete protein induction. I have to make the PBS wash steps and then cell lysis with glass beads and bead beater. Can I freeze the washed cell pellet at -80°C, resuspend in PBS and then make the lysis? Or freeze them in PBS (should I add glycerol or not)? I don't have liquid nitrogen or dry ice available, so I would have to freeze them in the -80 freezer.

Freeze the pellets in liquid nitrogen and store at -80 C. You can keep the pellets for years. For long term storage, avoid resuspension. Also, do not store as lysate or clarified lysate. These are ok for short term but not long term.

When you froze the pellet at -80ºC, is it still possible to have some protease activity from broken cells during the freezing process? 

Thank you