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Subcellular fractionation by centrifugation - Can u tell me a easy protocol? (Oct/12/2007 )

Hi! I'm starting with protein extraction (I've done in bacteria). I'm trying to identify how much of my protein is in the plasma membrane (most) and how much somewhere else in a eukaryotic cell line.

I would like to use the most simple method, avoiding steps like homogenization if possible, etc. I've seen that doing centrifugation at 1000g for 10minin the pressence of succrosse for density gradient my pellet should contain nuclei mitochondria, plasma membrane, and cell debris. And the supernatant the rest. I will detect it using luminescence emission of my protein (it has luciferase linked). Another question is: how I resuspend the pellet to solubilise the protein?

How I obtain the total protein of the cells (pellet, cytosol, everything) together? I want to do this to compare with the pellet light emission and the "cytosol" and other organelles light emission.

And one more question, if I want to get the proteins localised in the cytosol is good enough to centrifuge at 100000g for 45min, or I shoud go step by step, first 1000g to get rid of plama membrane, etc...then 3000g for 10min to eliminate mitochondria

This is one of the links I've been following:
http://www.axis-shield-density-gradient-me.../Applic/S07.pdf

Any suggestions, advice will be very appreciated. Also good links, papers or books with simple methods would be fantastic.

Thanks a lot.

-gsamsa-

QUOTE (gsamsa @ Oct 12 2007, 04:48 AM)
Hi! I'm starting with protein extraction (I've done in bacteria). I'm trying to identify how much of my protein is in the plasma membrane (most) and how much somewhere else in a eukaryotic cell line.

I would like to use the most simple method, avoiding steps like homogenization if possible, etc. I've seen that doing centrifugation at 1000g for 10minin the pressence of succrosse for density gradient my pellet should contain nuclei mitochondria, plasma membrane, and cell debris. And the supernatant the rest. I will detect it using luminescence emission of my protein (it has luciferase linked). Another question is: how I resuspend the pellet to solubilise the protein?

How I obtain the total protein of the cells (pellet, cytosol, everything) together? I want to do this to compare with the pellet light emission and the "cytosol" and other organelles light emission.

And one more question, if I want to get the proteins localised in the cytosol is good enough to centrifuge at 100000g for 45min, or I shoud go step by step, first 1000g to get rid of plama membrane, etc...then 3000g for 10min to eliminate mitochondria

This is one of the links I've been following:
http://www.axis-shield-density-gradient-me.../Applic/S07.pdf

Any suggestions, advice will be very appreciated. Also good links, papers or books with simple methods would be fantastic.

Thanks a lot.


a prerequisite to subfractionize cells is lhomogeneization or lysis by sonication or osmotic shock; uasge of detergenst is not to recommend; use sucrose density gradients; for some cells (blood cells f.i.) some companies offer ready-to-use gradient tubes

-The Bearer-

Thanks for the quick reply, that clarify some thoughts for me. I initially thought that homogeneization was a pre-requisite before doing the lysis. To do the lysis I was planning to use a hyposmotic solution (20mM Tris-HCl, 1mM NaEDTA, 5mM B-Mercaptoehtanol, pH=7.4). Open to suggestion about what buffers I should use (I prefer made by me rather than buying a commercial one)

-gsamsa-

QUOTE (gsamsa @ Oct 12 2007, 07:48 AM)
And one more question, if I want to get the proteins localised in the cytosol is good enough to centrifuge at 100000g for 45min, or I should go step by step, first 1000g to get rid of plama membrane, etc...then 3000g for 10min to eliminate mitochondria

you can just spin at 100000xg for 45-60 min to get the cytosol. no need to subfractionate if you don't need it.

-mdfenko-

I will do that then. Any advice about what buffer use to solubilise my protein, specially the pellet. Last time I used 20mM Tris-HCl, 1mM NaEDTA, 5mM B-Mercaptoehtanol, pH=7.4 but I don't think this solution was good to get the pellet in solution. Maybe changing the pH. What would you use?

-gsamsa-

the pellet will contain membranes and organelles. you may need to add detergent to the pellet to solubilize.

if all you want to do is to run some pellet on a gel (sds-page) then you could suspend some pellet in sample buffer.

-mdfenko-

Thanks once more.

I got a final doubt about the centrifugation protocol. Do I need to use Sucrose? Is it good enough the speed to separate membranes or I do need sucrose to get them separated?

-gsamsa-

we always prepare cytosol without sucrose. as long as you are not looking for subcellular fractions it should be okay without.

-mdfenko-