Membrane protein purification method ?

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Jason King
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Membrane protein purification method ?

I am interested to see whether a protein that is usually in the nucleus might also be associated with a membrane protein complex under certain conditions. I am looking for a reliable method that excludes nuclei to prevent fraction contamination issues. Does anyone have first hand experience with such a fractionation? I do not need the proteins to retain their activity - I just need to run the membrane fraction on an SDS-PAGE.
 
Thanks

Ivan Delgado
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Hi parvoman,
In my experience the easiest way to do this is by differential centrifugation. The nucleus (together with the plasma membrane) should pellet before anything else (500 xg for a few minutes). Membrane fractions require significantly stronger centrifugal forces to pellet (the ER will not come down until you spin at 50,000 xg for a solid hour or more). 
Alternatively you could also run gradients (like a sucrose gradient), but that takes significantly more work and optimization. 
Either case, you of course want to use good markers to make sure you are not having cross-contamination. 
Good luck

varsha
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I used to run the homogenate

I used to run the homogenate at 1000g to separate pellet and separate nuclei from the rest of the membranes and cytosol. As long as you have markers for nuclei to exclude cross-contamination, you would be good to go.
Varsha

Jason King
Jason King's picture
varsha wrote:

varsha wrote:

I used to run the homogenate at 1000g to separate pellet and separate nuclei from the rest of the membranes and cytosol. As long as you have markers for nuclei to exclude cross-contamination, you would be good to go.
Varsha

 
Which buffer did you use to resuspend the cells in prior to homogenization and which homogenization method did you use?
 
Thanks

Jason King
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Ivan wrote:

Ivan wrote:

 
Hi parvoman,
In my experience the easiest way to do this is by differential centrifugation. The nucleus (together with the plasma membrane) should pellet before anything else (500 xg for a few minutes). Membrane fractions require significantly stronger centrifugal forces to pellet (the ER will not come down until you spin at 50,000 xg for a solid hour or more). 
Alternatively you could also run gradients (like a sucrose gradient), but that takes significantly more work and optimization. 
Either case, you of course want to use good markers to make sure you are not having cross-contamination. 
Good luck

 
The nuclear extract method I have results in a pelleting of nuclei and plasma membranes together, so you just get rid of the cytoplasmic fraction. I did see some methods that involved various Triton X species used at specific temperatures to remove proteins from the plasma membrane. Has anyone tried any of these?

heehawmcduff
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I have used the following

I have used the following subcellular extraction kit with some degree of success.  You get fairly good purity and it is based on differential centrifugation together with using different combinations of extraction buffers.
http://www.merckbiosciences.co.uk/html/cbc/kitresource_proteinextraction_fractionation.htm
Hope that helps

coldmoon
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Hi, I have a similar question

Hi, I have a similar question here.
I have expressed a membrane protein in insect cells(sf9). I want to confirm that the protein is truly expressed on the membrane but not in an insoluble form in cytoplasm (which is possible). Is there any fast method to isolate pure membrane? Or what kind of centrifucation would seperate the insect membrane from the insoluble cytoplasmic fraction? I don't want to use detergents to solubilize membrane if I can't seperate it from insoluble proteins,because detergents might solubilize the latter too.
Anyone could help? I guess it might be really easy to someone in this field. Thanks!

g a
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Just in case you do not want

Just in case you do not want to use the detergents use a combination of UREA +THIOUREA in 7+2M however that will be if you want to analyze whole fraction. Incase you only want insect membrane fraction then It will be ideal to start with the hypotonic cell lysis solution, cells will lyse releasing the nuclei and cytoplasmic contents will be in the soup. Pellet the nuclei at 500g and then take the supernatant and add perform the density gradient centrifugation.
once you have the membrane fraction add 7+2M UREA+ THIOUREA and solubilize the membrane associated proteins with it.
thats where you achieve maximam solubilization of membrane proteins without involving use of a detergent.
 

varsha
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Hi Parvoman.

Hi Parvoman.
1) Homogeniztion followed by spin at 1000g
The homog. buffer I had used was:
20mM HEPES
5mM  MgCl2
2mM DTT
10% glycerol
30 strokes ina Dounce homogenizer (pistol B)
2) For TC cells: solubilize the cells using 1% NP40 in an isotonic buffer  -spin at 1000g for 1' at 4C
nuclear membrane is NP40 resistant, but other membranes are not.
 

Jason King
Jason King's picture
Coldmoon,

Coldmoon,

How about doing immunofluorescence on your SF9s. Grow them on glass coverslips or let them stick to slides with a poly-L-lysine coating, methanol fix after a brief re-hydration, use a primary Ab, wash then fluorescently congugated secondary Ab, wash and check under the 'scope. You might want to do confocal to be 100% sure of the plasma membrane localization.

Jason King
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HeehawMcDuff,

HeehawMcDuff,

I had a look at your link to the Merck products. The "ProteoExtract? Native Membrane Protein Extraction Kit" (M-PEK) looks like it might be even better suited to my needs than the S-PEK you have used. I'll have a closer look at each product's data sheet.

Thanks

PM

heehawmcduff
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Hi

Hi
Hope it works well.  You may also want to note that, in the kit that I used, it required pretty high cell numbers per extraction (I think around 10-20 million), so if you are doing several treatments, you'll need a fair number of flasks of cells.
Best of luck with it.  I'd be interested to know how you get on with it.

Jason King
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That shouldn't be a problem.

That shouldn't be a problem. My K562 cells grow like...