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Vesicle immunoprecipitation and Blue Native PAGE

vesicle immunoprecipitation Blue Native PAGE

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#1 flooff

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Posted 27 September 2012 - 05:52 AM

Hi all!

I want to look for native protein complexes in transport vesicles by Blue Native PAGE. I searched a lot on pubmed / the web for papers or protocolls in which Blue Native PAGE was performed after immunoprecipitation, but i didn't find anything. I've a protocoll to isolate complete vesicles by gradient centrifugation and then load the whole vesicle fraction on the Blue Native gel. But I'm interested in distinct vesicle population and therefore I want to perform vesicle immunoprecipitation of specific vesicle membrane proteins.

Has anybody experience in performing Blue Native PAGE of immunoprecipitated samples? Which amount of immunoprecipitate may I need for performing Blue Native PAGE (I use 6 10cm culture dishes for a normal vesicle gradient). How do I seperate the vesicle/antibody complex from ProteinA/G beads without denaturating the samples?! The protein complexes must remain in native conformation.

Any suggestions? Thank you very much!

Best regards,

Florian

#2 gmcstay

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Posted 29 September 2012 - 06:54 PM

Hello Florian,

I have been doing a very similar kind of thing using mitochondrial proteins that have been tagged with an epitope tag that can be eluted under native conditions. However, these are proteins that are introduced into cells and not the endogenous proteins themselves. You may be able to elute your protein with the peptide the antibody was raised against (if this is how the antibody was made).

As for the amount of protein to load, when you have an IPed sample you purify the sample so much that the amount of protein loaded on to the gel is really not a problem. I use 500ug of mitochondrial protein for my IP and when I do a western transfer of the gel I can see the two protein complexes stained by ponceau. BN-PAGE gels can resolve 500ug of mitochondrial protein fairly well, but I would routinely use 250ug for sharper bands. But an IP should not be a problem.

Hope this helps you out in some way,

Gavin





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