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Problem: protein precipitates from cell lysate after defrosting


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#1 Holsten

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Posted 17 March 2010 - 12:18 PM

Hey, guys.
I'm preparing cell lysates for IP of the transmembrane protein. I lyse cells for 30 min in this buffer: 1% NP40, 10 mM Tris HCl, pH 7.4, 150 mM NaCl, 1mM EDTA and proteases inhibitors. After lysis I spin down the debri and freeze the supernatant at -20.
The problem is that when I defrost the supernatant all the proteins seem to get precipitated. Why does freezing-thawing cause this precipitation? Any suggestions? Does anyone freeze cell lyzates before IP?

#2 mdfenko

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Posted 17 March 2010 - 12:38 PM

the proteins that are precipitating are denatured.

some proteins can't withstand freezing.

in this situation you can make the protein solution 50% glycerol and store at -20C. it won't freeze but will be stable for long periods.
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#3 Holsten

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Posted 17 March 2010 - 01:16 PM

Thank you for fast reply! 50% glycerol sounds good, but wouldn't it interfere with IP?

Edited by Holsten, 17 March 2010 - 01:16 PM.


#4 mdfenko

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Posted 26 March 2010 - 08:06 AM

Thank you for fast reply! 50% glycerol sounds good, but wouldn't it interfere with IP?

it shouldn't but you can dilute or dialyze to reduce the glycerol concentration.
talent does what it can
genius does what it must
i do what i get paid to do

#5 jejde

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Posted 24 September 2010 - 07:56 AM

I've recently had the same problem for older (1 month) cell lysates stored at -20C.
Can you recommend any way of re-introducing the denatured proteins into the solution (i.e. resolubilizing them)?
Also, why is it that once denatured they precipitate out of the solution?

Thanks!




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