ER membrane prep for 2D gels - liver micrsosomes (Dec/12/2007 )
The boss has put a request to me to figure out a way to run liver ER membranes on 2D gels.
What I will do is as follows:
Homogenize 100 mg mouse liver in a KPO4/ sucrose solution with protease inhibitors.
Spin 15min 13k xg to remove nuclear stuff and mitochondria.
Spin supernate 15 min 100kxg to pellet the ER membranes. The proteins in solution are cytosolic.
The cytosolic proteins I TCA ppt and Acetone wash and suspend in 7M urea, 2M thiourea, 4% CHAPS, 20mM DTT and run 350 ug on a 3-10 IEF strip and focus for 60,000 volt hours total. Run the strip on the slab and Cypro ruby red stain. It looks great.
I do the same thing with ER membrane proteins and it looks horrible. The proteins focus on the IEF strip, very little horizontal streaking, but in the vertical, they look like 'falling stars'. And there are very few proteins compared to the cytosolic fraction.
I tried to upload a pic file of it, but got told I am not allowed to upload this type of file. I'm not sure why, it is just a tiff file.
try converting the tiff file to a jpeg. that may work.
how are you preparing the ief strip for the second dimension? do you soak it in sds (and 2me or dtt)?
you may not be denaturing the proteins well enough prior to the second dimension.
The strip is soaked in the sample buffer containing the membrane proteins: i.e. urea, thiourea, chaps, DTT amphyolytes. I wouldn't want SDS as that is ionic.
Also, if this procedure is dentaturing the proteins well for the cytosolic proteins, why wouldn't it work for the membrane bround proteins?
I'll try the conversion, at the moment my computer refuses to recognize the jump drive the pic is on.
Damnit, that is exactly what it looks like on my mac too. But I emailed it to a guy with a pc and he saw it just fine.
Also, if this procedure is denaturing the proteins well for the cytosolic proteins, why wouldn't it work for the membrane bound proteins?
that is for the preparation of the ief strip. what do you do to the strip after ief (prior to 2nd dimension)? you would want sds at this stage.
membrane bound proteins are harder to work with than cytosolic proteins for a variety of reasons. they could be bound to lipids or other proteins. they could be less soluble at the conditions at which you are isolating them.
even though you seemed to separate them on ief, they may still be bound to other components of the membrane.
I think I got the situation worked out.
Suspend microsomes in 7M urea, 2M thiourea, 4%CHAPS, 2% Triton X-100. Rock 1hr room temp, then sonicate 6x10 second bursts, waiting 10 sec between bursts. Spin 10' 10kxg. I do have a small insoluable pellet which I will digest and do a modified lowery on.
Take 350ug of protein, bring to 450ul with same buffer as above, add DTT to 20mM, IPG buffer to 1%. Rock for 2hrs room them, then apply to IEF strip.
I will post pics once I figure out how to get them.