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co-IP after ubiquitin cleavage

Isopeptidase T Ubiquitin co-IP DTT

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#1 Wickerl3732

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Posted 14 September 2012 - 08:35 AM

Hi,
I am persuing a finding, that protein A interacts with protein B and that this interaction is either dependent of k48 ubiquitination of on of these proteins or dependent on unlinked ubiquitin chains. To verify my assumption, I wanted to digest the cell lysate with isopeptidase T to cleave the ubiquitin chains, and I expect that my two protein of interest would not co-immunoprecipitate anymore.
However, activation of isopeptitase requires 10mM DTT, and the enzymatic reaction should be performed with at least 1 mM DTT. I guess that this concentration of reducing agent will disrupt my protein-protein interaction anyway.

Does anyone have any suggestions how I could analyze the dependence of two proteins on ubiquitination? I would highly appreciate your help!

#2 mdfenko

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Posted 14 September 2012 - 11:32 AM

1mM may not affect your interaction (try it). we routinely maintain our proteins in 2mM dtt.

if it does prevent interaction then dialyze it out after cleavage (you can perform drop dialysis on a few ul).

Edited by mdfenko, 14 September 2012 - 11:35 AM.

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#3 proteaMatt

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Posted 18 September 2012 - 05:04 AM

This seems a like an interesting experiment that I could learn from, since I haven't done IP before. I don't know if you have access to MALDI-TOF, but that seems like the perfect way to analyze these proteins, depending on the condition of the sample (how "clean" it is) and the molecular weights of your POIs. It seems to me like you could perform IP on the your whole lysate, which would leave you with purified POIs. You could then follow that up with MALDI analysis (likely using Sinapic Acid as the matrix) with three samples - 1. the intact associated protiens, 2. disassociate the proteins but leave the ubiquitination intact (would be convenient if DTT could do this for you), 3. Isopeptitase enzymatically treated POIs.

If your hypothesis is true that ubiquitination contributes to the association, after MALDI you should know the masses of: 1. each POI without ubiquitin, 2. the ubiquitinated intact associated POIs, 3. the two POIs disassociated, which should allow you to determine which one retains the PTM due to the mass shift from #1

Maybe I'm totally off base with this, but that seems like it could work, if you don't have MALDI available you could try that with 2DE and Western Blot.

Edited by proteaMatt, 18 September 2012 - 05:05 AM.

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#4 Wickerl3732

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Posted 18 September 2012 - 05:30 AM

Hi, thanks for your answers and recommendations.

@mdfenko: in preliminary experiments in the reaction buffer containing 1 mM DTT the interaction was affected (in two out of 3 experiments). However, I'll try to dialyze after treatment in reaction buffer, but I'm not sure if the POIs will re-interact under non physiologic conditions.

@proteaMatt: this would actually be my plan...

But I guess my real question is if anyone has experience with ubiquitin cleaving enzymes. Maybe I could use a different peptidase rather than isopeptidase T (USP5) which would allow for different reaction buffers. I till would be happy for further recommendations.....

Thank you.

#5 proteaMatt

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Posted 18 September 2012 - 05:59 AM

Maybe I misunderstood your first post. But the difference between what I took as your suggested workflow and what I suggested is the point at which you do the IP. It seemed like you are concerned if Co-IP will still occur if you do the USP5 digestion on the whole lysate, so take the guess out of it and let the MALDI sort it out.

I guess the point I am making is this - if you aren't sure how the about the above workflow then just do 1. the Co-IP on the whole lysate (I am assuming you already know that your POIs Co-IP in their "intact" state), 2. apply treatments to the POIs (use whatever petidase you want here), 3. let the MALDI sort it out.

If you want to have a set of data to cross reference with the intact analysis I would suggest doing an in-solution tryptic digestion on the Co-IP samples and then analyze that by LC-MS, this could give you the exact modification sites of your POIs.

It seemed like you wanted to treat the Co-IP as sort of like an assay, and I am suggesting to treat it as a sample prep method and analyze with MALDI.

Edited by proteaMatt, 18 September 2012 - 06:06 AM.

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#6 Wickerl3732

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Posted 18 September 2012 - 06:22 AM

Hi proteaMatt.

now I got you! This indeed should work. Sorry that it took me a little bit longer to follow you...
Thanks





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