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Membrane protein isolation from postmortem brain tissue

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Dear all,

I wonder whether anyone can help!

I have been looking for a method (either purchase a kit or use an optimised protocol) which enables isolation of the membrane protein fraction from postmortem brain tissue, primarily the low abundance neurotransmitter receptors and other ion channels.

This method should also be compatible with the downstream LC-MS/MS analysis.

In fact, I have tried a few methods:

* Mem-Per eujaryotic membrane protein kit (Pierce)
* M-PEK (Calbiogen)
* S-PEK (Calbiogen)
* Conventional membrane protein isolation protocols based on ultracentrifugation and use of mild-detergents.

However, one of the problems that has always persisted was the fact that no matter what method is used there will always be cross-contamination of the membrane protein fraction by both cytosolic and cytoskeletal proteins.

So, does anyone have suggestions on how to overcome this problem or at least try to reduce cross-contamination?

Also, would anyone be able to suggest an optimal protocol for total membrane protein isolation and separation from the rest of the cellular proteins using brain tissue?


Thanks a lot!!
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