Message
From: Frank Niesen
To: CCP4BB@JISCMAIL.AC.UK
Sent: Friday, 23 April, 2010 10:40:59
Subject: Re: [ccp4bb] degradation of protein durring freez thaw
An aspect that is sometimes overlooked is that the need to avoid mini
freeze-thaw cycles does not only call for a quick, snap-freeze process
An aspect that is sometimes overlooked is that the need to avoid mini
freeze-thaw cycles does not only call for a quick, snap-freeze process for
samples (in thin-walled PCR tube and not too large volume; 50-100 ul I'd
recommend), but - in turn - also for quick thawing: I prefer holding the PCR
Lots of things change with temperature. An excellent book on the
subject is P. Dohzu's "Cryobiochemistry" (1977) Academic Press, which I
think every lab that freezes biological molecules should have on hand
($15 on Amazon.com). For example, did you know that the pH of a tris
buffer jumps from
A couple more thoughts:
1. thermodynamics says that proteins denature at low temperatures just as they
do at high temperatures.
2. flash-cooling does away with some of what thermodynamics says (not an
equilibrium process anymore)
3. Whether a given protein can be frozen needs to be experimentall
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 free
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
__
From: David Briggs [mailto:drdavidcbri...@gmail.com]
Sent: Thursday, 22 April 2010 6:07 PM
To: Peat, Tom (CMHT, Parkville)
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: RE: [ccp4bb] degradation of protein durring freez thaw
Ouch!
I completely agree re: single proteins, but I had always found/heard that
: [ccp4bb] degradation of protein durring freez thaw
Hi, Obvious answer - don't freeze it. If you cannot set your
crystallisation screens up straig...
@jiscmail.ac.uk] On Behalf Of David
Briggs
Sent: Thursday, 22 April 2010 5:27 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] degradation of protein durring freez thaw
Hi,
Obvious answer - don't freeze it. If you cannot set your crystallisation
screens up straight away (the preferred opt
Hi,
Obvious answer - don't freeze it. If you cannot set your crystallisation
screens up straight away (the preferred option, IMHO), why not try leaving
the protein at 4C overnight? Does it still degrade?
Freezing & thawing a protein-protein complex not a good idea, I think.
Hth,
Dave
--
Delive
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 enou
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