Re: IEF gels or strips fro 2D protein sequencing
Katheryn Resing (Katheryn.Resing@Colorado.EDU)
Fri, 16 Jan 1998 08:43:49 +0000
We are using immobilized IEF strips from pharamacia for 2Ds. We
chose this system because of the ability to load a lot of protein by
swelling the strips in the sample because we want to do mass spec on the
spots. After some initial problems involving proteolysis of the sample
during swelling and running, primarily showing up as absence of high
molecular weight proteins, we now get nice reproducibible 2Ds loading 0.5
mg total cell extract on the 4-7 pH range Pharmacia strips. We still see
proteolysis of the more sensitive of the large proteins, and are working on
that. We haven't tried scaling up, but I don't anticipate any difficulties
adding 4-5 X more protein, except that we will have to redesign our
extraction buffer (right now, we're diluting the sample into the suggested
solubilization buffer to minimize the salt that is in the extraction
buffer).
We have had problems with the 3-10 pH range strips, where some
strips give good results, but other strips show smearing at one end or the
other. As the proteins we are interested in are in the 4-7 pH range, we
have dropped the 3-10 strips for now.
We have seen no contaminants from the system, but we have been
using new oil each time and have a meticulous grad student doing the work.
In getting started, he had to call Pharmacia several times, and they were
generally quite helpful.
Katheryn Resing
> To all who run 2D gels:
>
> What do you think about using immobilized IEF strips for the 1st D of
> a 2 D gel for protein sequencing?
>
> Please comment on load, reproduciblity, ease of use and contaimates
> from the system.
>
>
> David T. Chin, Director
> UMC Protein Core
> e-mail: bcdchin@muccmail.missouri.edu
> (573)882-2027 (office and messaging)
> http://www.missouri.edu/~pc/