I'm running an assay that calls for use of trypsin to cleave a fluorescent peptide reporter, and the protocol specifies that trypsin (lipholized) should be aliquoted out at 300X working concentration and frozen at -80 for storage. The protocol does not include a resuspension buffer.

 

My question is, is it ok to just use DI water to reconstitute the solid trypsin that I have and then freeze at -80? Most of the literature says that trypsin life can be lengthened by first dropping the solution pH with either 1mM HCl or 50mM acetic acid to reversibly inactivate the trypsin so it doesn't autolyse itself during storage at 4 or -20. I can't find anything about -80 storage however. Is the activity sufficiently curbed at this temperature that acid does not need to be added to prevent autolysis during storage? I don't want to introduce extra acid to one of my assay components if I don't need to.

 

Thanks!

 

Generally I prefer to store trypsin dry whenever possible and only reconstitute the amount I need per use. I cannot tell you with certainty about stability in water, but one thing to keep in mind is that unless you flash freeze it, it takes quite a while to reach -80 and that there is quite a bit of time needed for that to thaw up again.

That being said, for cell culture trypsinization, trypsin solution is routinely frozen at -20 to -80 C in an EDTA-saline solution. So if kinetics and a bit of loss is not a huge issue, I think it should be fine. I would probably still use very small aliquots to minimize the time to freeze/thaw.

Generally I prefer to store trypsin dry whenever possible and only reconstitute the amount I need per use. I cannot tell you with certainty about stability in water, but one thing to keep in mind is that unless you flash freeze it, it takes quite a while to reach -80 and that there is quite a bit of time needed for that to thaw up again.

That being said, for cell culture trypsinization, trypsin solution is routinely frozen at -20 to -80 C in an EDTA-saline solution. So if kinetics and a bit of loss is not a huge issue, I think it should be fine. I would probably still use very small aliquots to minimize the time to freeze/thaw.

 

CharonY,

 

Thanks! Do you think that aliquots of 500ul would be a problem? As I understand it you want to reduce the amount of time during freeze/thaw to prevent compromising activity?

 

Thanks!

Yes that is the basic idea. I do not have actual quantitative values to support it, it is more a feel-good-lab-procedure (like the occasional blood sacrifice to keep the PCR gods happy).

 

Personally, I would aim for a volume that corresponds to a single-use. If it is 500ul in your case, I would aim for that. It should be noted that storage time should not be considered indefinite. While I have seen aliquots still active after a few months, the efficiency can be quite variable past a month or so. I suspect that in some case at least it is also a matter of how cleanly you prepared your aliquots.

Hi! do you think is it ok to store trypsin in ABC for a month in -80? I am using it for pulldown experiments..

Thank you

Maria