Ubiquitination

Share protocols and ask for sample preparation advice.
pdow01
Carbon Member
Carbon Member
Posts: 3
Joined: Thu Jun 05, 2014 1:20 am

Ubiquitination

Postby pdow01 » Mon Jun 09, 2014 1:07 am

Hi All,

I am pulling down a protein from a cell lysate using immunoprecipitation. I intend to run this on a gel, cut out some gel segments from the molecular weight of the proteins upwards, digest and analyse by mass spec. I want to try and identify potential ubiquitination sites on my protein of interest. Does anyone have any experience of this protocol and is there any thing I need to be aware of?

Kind Regards,

Paul.

Infinity
E. Coli Lysate Member
E. Coli Lysate Member
Posts: 107
Joined: Wed Dec 21, 2011 8:22 pm

Postby Infinity » Mon Jun 09, 2014 8:46 am

Well, first do not expect that after IP you will end up with 100% ub-proteins, in fact probably it won't work at all (w/o considerable optimization efforts), second - even if you have ub-protein you might not detect corresponding ub-peptide since protein coverage is typically much < 100%. My guess is that after this experiment you will see a lot of albumin, keratin and other high abundant proteins. In any case before doing MS I would recommend to estimate efficiency of your IP by WB.


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