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Identification of Membrane Receptor protein, how ??


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#1 marcos.brazil

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Posted 16 February 2011 - 03:46 AM

Hi everybody !

I work with a secreted protein that we know that it have a membrane receptor ! So we want to identify "who" is this receptor.

But I have no experience on membrane proteins and further questions

What is the best approach that you sugest for me ?

I am thinking in incubate the recombinant protein (Q0 - How much protein ?) with a cell culture, at 4oC (Q1- best temperature?),wash a lot with PBS, and cross-link it (Q2 - how?)

after cross-linking I imagine to incubate with the antibody against my protein, wash again, and the critical point isolate the complex. First I imagine that i have to lysis the cell (Q3 - how ?), isolate the membrane fraction (Q4 - ultracentrifugation ?), solubilize it (Q5 - best buffer that dont disrupt the antibody binding to the protein ?), immunoprecipitate it with G protein beads, SDSPAGE without desnaturant buffer and WB. So I will know the size of the band where is the complex my protein-receptor cross linked, and finally SDSPAGE with silver staining for MS identification

What do you think about this strategy ? I have either a monoclonal antibody for my protein biothynilated...

can you give me ideas and help me ??

thanks a lot

Marcos

#2 proteaMatt

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Posted 16 February 2011 - 07:36 AM

and finally SDSPAGE with silver staining for MS identification


What sort of MS are you prepping your sample for? I know that silver stain can cause problems for MALDI-TOF/TOF
Lab Technician at Protea Biosciences

#3 marcos.brazil

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Posted 16 February 2011 - 08:05 AM

yeah, I will destaining it with th kit, by the way, I forgot to write

and I really dont know so much about MS, so for this porpouse, which one do you suggest ?

#4 scolix

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Posted 16 February 2011 - 10:55 AM

another way to do it.

Try to make the secreted protein with His tag and then incubate protein with cell lysate and purify with nickel column and send sample for protein sequencing.

#5 marcos.brazil

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Posted 16 February 2011 - 12:01 PM

Tks !!

in fact my protein has the his tag, but niquel/colbalt affinity is not that specific, so propably can give me false positives !!

or I can try a tandem purification, that could be great !

Thanks !




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