membrane protein extraction

Hi! I need to demonstrate that my protein of interest is really present in the membrane. I would like to prepare protein extracts separating the fraction of the membrane-bound protein from the cytoplasmic/nuclear ones, so that I could do an immunoblotting on the two different fractions. I work on cell culture, not on tissues.

My standard protocol for protein extraction allow me to obtain total extracts, using this buffer:

 Hepes pH7.5 50 mM, NaCl 150 mM, glycerol 10%, Triton x 100 1%, MgCl 1.5mM, EGTA pH8 5 mM, PMSF 4 mM, aprotinin 4%, Sodium orthovanadate 10 mM, sodio pyrophosphate 20 mM.

Then after centrifugation @ 13000rpm I discard the pellet and load the sample on SDS-PAGE.

 Can anyone suggest me a solution to my problem?

Thank you!

Betta

I did not really get your question.

Do you mean that you need to show the efficiency of your blotting? if that, the simple to do it is to stain your membrane with Ponceau. that tells you if your proteins were blotted. Also stain your gel after blotting, this tells you if all your proteins were blotted on the membrane.

let me know if it helps

Good luck

Well, probably my question was not clear: I referred to the plasmatic membrane, not to the blotting membrane. The problem for me is to discriminate the proteins residing in the cytoplams from the proteins expressed in the plasmatic membrane.

But thankyou for your effort!

Betta

I think Betta wants to do a membrane prep to determine whether her protein in the cell membrane.

I used to do this by a 3 step prep that involved 2 ultracentrifugation preps, quite time consuming. However there are now a few great kits on the market that do this pretty easily

Here's one from Qiagen - http://www1.qiagen.com/products/catalog.aspx?productlineid=1000625&

or this one from Pierce (Thermo) www.piercenet.com/Objects/View.cfm

If you dont want to buy and try this kits then, I will try to dig up my old membrane prep protocol for you.

If you do please let us know how they worked for you.

Best

Rusty

Sorry, after reading it agin, now seems that I got your question.

If I am right, you are planning some work on membrane protein, and you need to show that protein is in membrane fraction, while not in soluble fraction.

So, I will suggest

1. Extract your cell culture with your extraction buffer (without detergent, which will extract your membrane protein). The original ectraction buffer you listed is for total protein.


2. Spin down at a lower speed (the speed you used to spin down your cells, I am using 1800g for my cells) to get rid of any un-brooken cells and cell debris


3. Take supernatant, and spin down at ~60,000g if you have a ultra-speed centrifuge. If not, spin down at the max speed with a table top centrifuge


4. Take supernatant, which is your soluble protein fraction


5. Resuspend the pellet with your extraction buffer + detergent (I will suggest 2% instead of 1%). Or simply resuspend the pellet with 1X SDS sample buffer


6. Follow your SDS-PAGE protocol, and then western blot

Good Luck

Rusty, You get the question!

Since I need to do it just in this particular case, well, the commercial and rather expensive kits are my last chance..... Thankyou for your protol, I will let you know if it works.

In the meantime, well, I'm really grateful to you....

Betta

I think the post above is a reasonable approximation of what I would give you for a protocol, so try that first.

Your welcome. This is what we do!  Tell your labmates and friends please.

Rusty

Hi Betta,

If you would like more information about how to separate membranes, take a look at this protocol:

http://www.ivaan.com/protocols/122.html

This is the one I used to separate various membranes, as described in this publication:

http://www.plantphysiol.org/cgi/content/full/116/4/1339

In short, different speeds separate different membranes. The experiment can be simple if you are only looking to determine membrane-bound versus soluble, and more complicated if you want to be more specific as far as which membrane your protein is associated with. 

Good luck