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" <[email protected]>:
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
