I have to agree that protein is denaturing or precipitating irreversibly during the freeze-thaw cycle. While HOW you freeze does matter (cooling slowly in a freezer is almost always destined to fail) it should be noted that many proteins simply won't tolerate freezing. (I seem to specialize in freeze-intolerant proteins for some reason.)
My first inkling would be to try stabilizing the protein at 4C which is often possible. For proteins susceptible to oxidation, the biggest enemies are oxygen and trace metal ions. A minimum amount of reducing agent and chelator does wonders for 4C stability. (Glycerol is great, too but usually interferes with crystallization.) It's worth a try. (Some proteins aren't stable at 4C either. I know a lot of those proteins, too.) Cheers, Roger Rowlett On 4/22/10, Shao-Yang Ku <s...@embl-hamburg.de> wrote: > From your description, the protein concentration dropped from 10mg/mL > to 1-2mg/mL after freeze-thaw. It's hard to imagine that your protein > has been degraded. Degraded by what (protease)? At -80oC? Did you see > a ladder of lower bands on the gel after the freeze-thaw? > > If you're interested in anecdote, a protein I used to work on would > degrade at 4oC (but in 2 days, not overnight) and precipitate (after > thawing) when snap-frozen in liquid nitrogen unless there is at least > 25% glycerol in the buffer. > > Good Luck, > SY > > Quoting "Jhon Thomas" <jhon1.tho...@gmail.com>: > >> Hello BB >> >> I apolozize an off topic query. >> >> I am working with small proetin-protein complex of 24kDa. I purify this >> N-terminal His-tagged complex through tylon resin in 20mM of Tris pH-8.0, >> 0.3M NaCl . After purification this protein complex are dialysed in 20mM >> tris pH=8.0.I am able to purify enough amount of protein for >> crystallisation, which can be concentrated upto 10mg per ml. Then i check >> the dgradation on the polyacrylamide gel after concentration of the >> protein. >> I donot see any degdation protein band on the gel. I store the protein at >> -80 in aliquotes of 100ul immedaitely after concentration in same buffer. >> protein concentartion are done at 4 degree by centrifugation. Next day >> before setting up the trays for crystallisation screening, protein >> solution >> concentration check is being done. it turns out that this complex has >> degraded and concentration is only 1-2 mg per ml. i would appreciate the >> suggestions to prevent the degradation of complex or How should i make it >> more stable? so, that i can proceed for the crystallisation. I would >> really >> appreciate the suggestions. >> >> >> Thanks in advance >> >> Thomas >> > -- Sent from my mobile device
