Immunoprecipitated samples??

Ian A. York iayork at panix.com
Mon Aug 21 14:32:44 EST 2000


[Posted and mailed]

In article <Pine.A41.4.10.10008211256010.89696-100000 at acs6.acs.ucalgary.ca>,
Neal Robert Melvin  <nrmelvin at ucalgary.ca> wrote:
>I was wondering if anyone routinely freezes IP samples after the
>procedure... say in instances where you've finished the IP, but don't have
>time to run it on a gel... I assume this is probably an antigen-dependent
>thing, but does anyone have any insights?? Is it best to carry it as far
>as denaturing (95 degrees for 5 mins) before freezing?

I often do it, but I can't say I've done much in the way of comparisons.  
If I'm going to run the sample the next day, I usually denature and stick
it at 4 oC overnight; I reason that the freeze-thaw is more likely to be
harmful than any proteases remaining after the denaturing.  If I'm going
to wait a few days (or, more typically, if I've run the same and I suspect
I'll repeat the run for some reason) I'll store it at -20 oC, unless
experience has told me that the antigen is fragile.  If it does seem
fragile, or if I expect to wait for more than a week, I store it at -80
oC.  

As I say, my reasoning on these is basically guesswork, but at least for a
handful of proteins repeating runs after overnight 4 oC has given very
similar results.  I've seen degradation of antigen after -20 oC storage
for a few weeks, though these samples were being frozen and thawed
repeatedly.  I haven't seen a problem with -80 oC storage, and I'd
recommend this if your samples are precious.

The best option is running the sample at once, and I think it's worth
staying up late to get them on a gel.

Ian 
-- 
    Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
    "-but as he was a York, I am rather inclined to suppose him a
     very respectable Man." -Jane Austen, The History of England






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