[batty2279]

I know that when freezing proteins in the -80 C freezer, they should only be thawed once, or risk losing functionality.

My question is are the proteins more stable when still inside the cell... as in I had a batch of cells that I froze at -80, thawed once to lyse, then re-froze the protein supernatant down.  Technically the proteins from those cells have been frozen twice (thawed once), but could the stability of the proteins been kept intact since they hadn't been lysed yet?

I know it's an odd question, but I have a ton of protein lysates I want to use from over a year ago in the -80 and am concerned about their viability.  They were frozen down with PMSF/PIC, as well as with glycerol.  Any insight is appreciated.

[Babcock_Hall]

Naturally one wishes to minimize the number of freeze-thaw cycles, but IMO the question of whether or not activity is maintained has to be determined in a case-by-case manner.  If you have an assay for function, then you can assess it under the conditions you have.  There are at least three main variables.  One is the protein itself, two is the speed of freezing, and the three is the presence or absence of cryoprotective agents during the freezing or thawing process.  Sometimes substances such as glycerol, sucrose, trehalose, or other compounds (not always polyalcohols) are able to prevent damage.

[yesway]

Hi,

from my experience, It shouldn't be that bad, especially as the supernatant was re-frozen in glycerol (what conc?). I normally would not count collected & frozen cells after expression as a freeze/thaw cycle. However, you should expect some loss, but not substantial (your mileage may vary from protein to protein).