Can membrane proteins be separated using 2D-PAGE?!?! - (Jun/29/2007 )

Hey Guys! I'm having a few problems with 2D-PAGE gels and I was wondering if anyone had any suggestions for how to overcome them please?

I am preparing proteins derived from the cell surface of pig oviductal epithelial cells, with the idea of doing 2D-PAGE on them. I'm aware that as they are membrane proteins it is notoriously difficult to get them to run on 2D-PAGE. However, my boss is adamant that it can be done, although it maybe troublesome.

First of all I am having problems getting a high recovery rate after protein precipitation (I am using the GE Healthcare 2D clean up kit). It works out I am getting approx. ~35% recovery. Does anyone have any suggestions for how to increase this? Is there another method/kit available that gives a better recovery?

Secondly, I have started running some of my samples on the first dimension. They run ok up until they reach 8000v, but then the strips start coming out of the mineral oil and I assume possibly begin to burn and don't run properly. I've tried to over come this by stopping the program and adding more mineral oil to the wells, but this doesn't stop the strips coming out of the mineral oil - they still appear to quiver in the wells. Does anyone have any suggestions for how to overcome this? Or is this project doomed from the start?!?!?!

I've read a couple of papers where people have got results for cell surface proteins using 2D-PAGE, however, none of them go into the problems they would have had getting the gels to run well!

Any help anyone has would be really appreciated as I'm really struggling with what to do to get this to work!

Thanks for your help guys! Hope you have a really good weekend xx

in deed, membrane proteins, especially those >100 kDa are more difficult in 2D SDS PAGE than f.i. cytosolics;

we use Invitrogen Zoom/NuPage system and very satisfied although it is not useful to study 1000 of proteins; Invitrogen offers 2 different Protein solubilizer for hardly soluble proteins; that should be worked out which is better of the two; it sholud be compatible with Amersham/Multiphore system

Hi!

Getting your membrane proteins on to your gels isnt easy. It takes a lot of troubleshooting to get it right. As far as precipitation goes, we usually use standard methanol/chloroform precipitations, or acetone precipitations. We use the MeOH/CH3Cl precipitations if we suspect that there is a lot of salt in the sample.

Can you tell us more about your problems and conditions? What is actually happening to your strips? How much salt is in your samples? You can buy small hand-held conductivity meters to measure how salty your samples are. What about protein load? Is this OK for your strip size? Are your strips blocking (forming white precipitated regions in the IPG strips)?