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Western for membrane bound receptor


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#1 dld2002

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Posted 25 January 2005 - 09:58 AM

I would like to perform a western to look at the levels of a specific receptor but I am not sure what would be the best method to extract proteins. I am afraid I will lose the receptor in my centrifugation step if I do not extract it from the membrane with a buffer more stingent than RIPA.

I would appreciate any ideas.

Thanks,
Dan

#2 littlecell

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Posted 26 January 2005 - 02:21 PM

i ever extracted one GPCR(membrane protein) from rat brain with a very simple buffer containing 5 mM Tris-HCl, pH 7.5, 5 mM EDTA, 5 mM EGTA. tissues were homogenized with dounce glass homogenizer on ice in the presence of PMSF. then the sample was centrifuged at 3000rpm for 10 min at 4 centigrade. collect the supernant (it's the membrane extract) and spin the membrane extract down at> 20000rpm for 30min at 4 centigrade and you will get the membrane protein extract, wash it once with TRIS. it's pure enough for WESTERN BLOT. that's what i did before.




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