Rapigest for elution during IP

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StephenLiverpool
Carbon Member
Carbon Member
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Joined: Tue Apr 29, 2014 2:15 am

Rapigest for elution during IP

Postby StephenLiverpool » Mon Dec 08, 2014 10:18 am

Hi everyone,

I'm looking for a MS-compatible approach to elute immunoprecipitated proteins prior to proteolysis. Does anyone have experience with using Rapigest (or similar acid-labile surfactants) for this purpose? Ideally, I would like to obtain quantitative recovery - what sort of concentration would be required to achieve this?

Many thanks,
Stephen

RDUnwin
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Joined: Sun Jul 24, 2011 11:29 pm

Postby RDUnwin » Tue Feb 24, 2015 4:31 am

We've done this successfully with 1% Rapigest. Worked really well. We never checked %age recovery, although if we then boiled the beads in Laemlli buffer we couldn't get more off. Also replicate ipps appeared reproducible....

Best
Richard

Christopher
Angiotensin Member
Angiotensin Member
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Joined: Thu Dec 27, 2012 12:26 pm

Postby Christopher » Tue Mar 03, 2015 6:53 am

We elute our IPs in Laemlli buffer and then clean them up prior to proteolysis using SP3 (http://www.ncbi.nlm.nih.gov/pubmed/25358341). You can use whatever version of Laemlli that you want, with or without glycerol, with our without dye. We also elute in RIPA sometimes. SP3 will get rid of whatever components you use in your elution. Then you can go straight to MS after your digestion without having to clean up the Rapigest.


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