We want to use 2D to identify phosphorylations on proteins of 90 to 150kDa. We have a BioRad 2D electrophoresis system that uses store bought strips for running the IEF (first dimension). We found out that no commercial strips will resolve proteins of the size that we are interested in. Anyone have any successful experiences in resolving large size proteins by 2D ?

Hi There,
You shold not have any problem I used to run on BioRad IEF stips pH3-10 and resolved a 100kDa protein. Also looked for phosphorilation using ProQ Diamond. So Try to do it once. If you still encounter problems let me know and we will try to fix it.
Guy

we've tried for months, different running times, etc... We recently did a silver stain and couldn't see proteins above 75kD on the 2D gel.
We asked technical support at BioRad and they basically said no commercial strips resolve proteins >100kDa.
If you were able to do this, can you send me a protocol of how you load your sample (how much protein?) onto the strip and your running conditions for the IEF ? Much thanks.
We are running whole cell lysates of Hela cells.

That is quite interesting.
First of all try staining with SyproRuby stain much better. Silver staining is not as sensative.
I used the same protocol that is described by BioRad
here is the system that I have used:
PROTEAN IEF Cell
With the 11cm strips.
I used just membrane lysosomes from rat hepatocyte.
I pre cleaned the sample with biorad precleaning kit.
As I said I used the protocol that was described in the BioRad booklet that was provided by them.
I loaded 75ug of total protein.
look at that link:
http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16917501
At fig 3A you will see one of my 2D you can defenetly see protein ranging pI 4-10 at the 100kD MW.

I guess that you either have problem of sample preparation or staining.
Did you do a regular SDS to see the protein of intrest ?
Guy