IP and 2D gel analysis - buffer composition (Sep/22/2005 )

I have IP conditions worked out for a protein of interest of mine, seems to work really well. We're now interested in phospho-mapping it, and IDing interacting proteins by 2Dgel ms/ms.

i've heard that the 1st dimension isoelectric focusing is very intolerant of salts, detergents, etc, of which there is alot in my IP buffer. do i need to reoptimize my IPs using different buffers, or can i do a low-salt wash prior to boiling the beads in urea, or is this not even something to worry about?
thanks in advance!

Hello,

1st dimension IEF isn't tolerant of salts in the buffer...you'll get poor resolution in your separation, most likely due to an unnecessary increase in temperature during focusing. There are ways around this though, without having to worry about changing your IP elution conditions. The most simple way is to simply perform a TCA or acetone precipitation on your eluate and re-suspened in sample buffer. Additionally, Amersham (or whatever they're calling themselves this week) sells a 2D cleanup kit that works well. Another alternative is to do a mini-dialysis in a microfuge tube. This technique is quite common in labs that frequently perform conventional IEF. I think Spectrum sells products for this.

If you like, you can email me your protocol and I can tell you if you even need to worry about this.

Good luck,

Jon
jonmike.reed@gmail.com