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isolation of membrane associated proteins


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#1 ndeweerd

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Posted 05 August 2009 - 09:44 PM

Dear All,
I am looking for a (reliable) method for the isolation of membrane associated proteins and cell fractionation. I would like to lyse my cells (stably transfected MEF's) without dissolving the membranes, spin down the membrane-associated proteins and then use Western blotting to discover whether the transfected receptor is present on the cell surface (membranes), in the cytoplasm or in subcellular organelles.

Can anyone help?

Nicky

#2 AVEA

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Posted 06 August 2009 - 05:45 AM

Dear All,
I am looking for a (reliable) method for the isolation of membrane associated proteins and cell fractionation. I would like to lyse my cells (stably transfected MEF's) without dissolving the membranes, spin down the membrane-associated proteins and then use Western blotting to discover whether the transfected receptor is present on the cell surface (membranes), in the cytoplasm or in subcellular organelles.

Can anyone help?

Nicky


There are two really good products that I know of that work to extract membrane associated proteins. The first is Mem-PER http://www.piercenet...WT.mc_id=keynum which will extract Cytoplasmic and Membrane proteins. The other one is the Subcellular Protein Fractionation Kit http://www.piercenet...WT.mc_id=keynum which extracts cytoplasmic, nuclear, membrane, chromatin bound, and cytoskeletal proteins. Both of the kits work very well and can be used with Western Blot. For your particular question the Subcelluar kit might be the best.

#3 D.Huckab

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Posted 07 August 2009 - 08:08 AM

Those kits that AVEA posted look really nice! If you are looking for something quick you can also do a detergent fractionation. Digitonin is commonly used and I have used it with good success.

My general protocol:
To extract a cytoplasmic fraction, first make a lysis buffer that has a very low concentration of detergent (40 ug/ml) and load with cells for a short amount of time (2 min). Remove and retain sup and load a higher concentration of detergent to extract membrane proteins. I have used 1% digitonin for 30 minutes for this step. All steps are performed at 4 degrees C, rocking.

For reference, I use primary CGN's attached to a bromosilicate plate, at a density of ~4 million cells.




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