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How to rescue my proteins?

proteins precipitation solubilization

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#1 Nephrite

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Posted 04 May 2012 - 10:32 AM

Hello.

I am in a big trouble.

The story:
I precipitated my crude extract of skeletal muscle proteins with ice-cold acetone and then solubilized them in buffer containing 8M Urea and 66 mM DTT as I did in the past.
The original protocol requires also CHAPS but here we don`t have.
In the past I used a powerful sonicator to dissolve the pellet but our new sonicator is very weak - I don`t know if this matters.

The problem:
Somehow I have been overdilluted my samples - the Bradford did not detect the amount in work dilution and the Urea in the stock protein solution was of incompatible concentration. Even undilluted portion reulted in pale staining of Bradford. I tryed protein estimation at 280nm as an alternative (vs BSA standards) - the absorptions were frustrating.
We don`t have any concentrators, dialysers, etc. - nobody here works with proteins.
So, today I decided to re-sediment the samples again in acetone and to resuspend them in less volume of 3 times dilluted Urea buffer. Some part of the proteins was dissolved but the main amount is like clumps on the bottom. Nothing helped. Well, the Bradford detected some protein amount but some of my proteins of interested are membrane-bound and I am afraid they are arrested in the clumps.

My questions:
1. Is there any way to rescue these samples, which are precious?
2. If I have to repeat how could avoid this situation - may I use Triton x100 instead of CHAPS?

Thank you.

#2 mdfenko

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Posted 07 May 2012 - 10:45 AM

you can try homogenizing the clumps in your urea-dtt buffer with a dounce.

triton (non-ionic) is not a replacement for chaps (zwitterionic). it might work, anyway.
talent does what it can
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#3 Nephrite

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Posted 08 May 2012 - 06:35 AM

Thank you! :-)





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