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" <[log in to unmask]>:
> 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
>
|