2nd dimension transfer problem

[kasta]

Hi,

I have begun 2D work on human fluids. I keep running into a problem of some proteins not entering the 2nd dimension.

In this picture i ran 10ug of protein in rehydration buffer (20mM tris, 8M urea, 4%CHAPS 0.5% ampholyte (3-10) and 50mM DTT) (overnight 14hours)

Total focussing was for 25kVhr on a 7cm pH3-10 IPG.

Second dimension equilibrated 1) in DTT and 2x in iodoacetamide) the SDS-PAGE on 8-16% tris-hcl gel.

Possibly they are some high MW proteins? or is there something i might be forgetting?

thanks,

[kasta]

* they are actually entering the stacking gel (4%) so maybe they just are high MW proteins...

[proteaGreg]

Kasta,
I'm a lab technician at Protea Biosciences and have experience running human fluids on 2D gels and judging from the image of your gel that you posted I think the problems you are having with your gel are coming from the high amount of albumin present in your sample. Protea offers sample prep kits for the quick and efficient removal of albumin form samples. Once the albumin is removed from the sample your gel should run much better. If you would like more information on our albumin removal sample prep kits please visit : proteabio.com/products/SP-200
I hope this helps!