Re: [ccp4bb] Acrylamide in RNA crystallization

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: Re: Acrylamide in RNA crystallization
From: Pascal Egea pascal {- at -} MSG {- dot -} UCSF {- dot -} EDU
Date: 2009-04-02

Hi Vanessa
It is better to get rid of traces of residual acrylamide that may
contaminate your final purified and refolded RNA.
It is usual to have contaminations with monomeric acrylamide. NMR
spectroscopists studying RNA can usually detect its presence on their
spectra.
If you can dialyze your purified product to try to get rid of it it would be
the best. Traces (sometimes it is not a negligible amount) are not good
because you may not be able to reproduce your results and optimize eventual
crystals. And for RNA this can be an excruciating pain.
When we transcribe RNA, we usually run the preparative acrylamide-urea gels
and elute the RNA out of the gel (most of the time by electroelution). The
RNA usually contains urea and acrylamide so I either precipitate using the
salt/ethanol technique and then resuspend the pellet and dialyze/refold or I
further purifiy on an ion exchange (Q type column) to try to clean it up.

If you have an NMR spectroscopist friend around, try to look at the presence
of acrylamide before and after these steps and see what works the best for
you.

Hope this helps

Pascal Egea, PhD
University of California San Francisco
Department of Biochemistry and Biophysics

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: RIP SGI
From: "William G {- dot -} Scott" wgscott {- at -} CHEMISTRY {- dot -} UCSC {- dot -} EDU
Date: 2009-04-01

Next message:
Subject: ligand refinemnet
From: peter hudson peter {- dot -} hudson {- dot -} peter {- at -} GMAIL {- dot -} COM
Date: 2009-04-02