Membrane protein western blot

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William31
William31's picture
Membrane protein western blot

Hello everyone,

I have read somewhere that membrane proteins do NOT like to be boiled before loading in SDS PAGE (
Lamelli sample buffer used (161-0737)).

Indeed, I am working with a membrane protein and I don't get any signal at all when I load in SDS PAGE and then I transfer proteins from the gel to PVDF membrane (semi dry transfer). However, if I blot the proteins directly into PVDF membrane I got a signal (chemiluminescent and flurorescent substrats).

Maybe the problem is the protein aggregation because they are boiled, so thay can't migrate very well in the gel or the transfer is not efficient.

But I don't boil the proteins, how can I prevent any protease activity ? By protease inhibitors adding ?

I am gonna try with different treatment like 70°C, 90°C and no heating/boiling at all.

Thanks for your help !

protoldo
protoldo's picture
Bonjour, the idea of don´t

Bonjour, the idea of don´t boil membrane proteins is to see the original conformation , but is necessary a loading solution without reducer. For instance for the chemoquines receptor you isolate your membrane fraction and take a sample for PAGE add the corresponding loading sol. with SDS but without reducer and apply without boil. SDS envelopes the structures in order to separate them by size and you can see associations multimerization etc. Good luck