I am trying to store my total protein isolated from cell lines. This is to be stored until I get my slot to perform relative protein profiling using LC-MS. Which would be the ideal method to store thye isolated protein (-80/ trypsinize and store at 4 c). Please shed some light over it.
Thank you
There are two options I'd recommend - either store cell pellets at -80, or extract, digest, cleanup and dry peptides and store them try at -20.
However, if for some reasons you want to store undigested proteins after lysis, don't forget to add protease inhibitors and keep them at -80.
Depending on the length of time you have to wait ! some labs store the peptides directly on the C18 material or stage tips, as long as they dont dry out.
@ Alexey L Chernobrovkin
On storage of protein extracts. You recommend to add protease inhibitors. Are there still proteases active at -80°C? And how would you then remove these inhibitors to be able to do the tryptic digestion? Or isn't this necessary because of the high concentration of Trypsin added?
Dear Ronny Mohren,
I doubt that any endogenous proteases are still active at -80. But protein degradation can happen before your samples are frozen, or right after unfreezing.
Protease inhibitor cocktails we use normally do not affect trypsin activity, mainly because sample prep involves protein precipitation prior digestion, and thermal or chemical denaturation.
Hi Balaji,
It is best to keep the samples, before lysis, at minus 80. When you are ready to carry out your digestion, lyse the materials with protease inhibitors and carry out the trypsin digestion.
Next day, purify the peptides over SepPak tC18 cartridges. The eluted peptides will be in high organic solvent, 70-80% acetonitrile with 0.1% TFA. This will not freeze at minus 20 and you should store your peptide solutions at minus 80 degree Celsius. Just before LC-MS/MS, speed vac the peptide solutions and reconstitute the material in 0.1% formic acid.
Good luck,
Hediye.
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