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Isoelectric focusing - Bands r curves.. any ideas to make them straight? (Sep/15/2006 )

Hi all,

I am doing a horizontal Isoelectric focusing gel, see some 6 isoforms with a method.. 8.5-10 pI gradient, 5% gel with urea.. 1.5 hrs (200V) prefocusing.. sample loading and then 1.5 hours focusing (400 V). protein pI ~9.4.

But my bands r constant curves (that's how my boss put it;)).. any ideas to make them straight?

Any reasons for such a thing?

Waiting for quick answers, Thanks!

-cheeztoast-

the curving may be caused by proximity to the electrode buffer strip (you should be able to see distortion waves originating at the strip). you can reduce the distortion by blotting your strips so that they are not too moist.

if you are not using buffer strips then disregard the previous part of this post.

you might also be able to reduce distortion by adjusting the range of your gel. try to make the range of your gel so that your protein is closer to the center of the pH gradient. in your case you will need to run a (very) high pH range gel.

-mdfenko-

Hi cheeztoast

I see that you had some problem with bands that were diffused. You were asking if the higher voltage of a longer run time will avoid this. I am in face of the same problem. Do you get in the meanwhile an answere to this question?

thanks
cuca

QUOTE (mdfenko @ Sep 15 2006, 10:16 PM)
the curving may be caused by proximity to the electrode buffer strip (you should be able to see distortion waves originating at the strip). you can reduce the distortion by blotting your strips so that they are not too moist.

if you are not using buffer strips then disregard the previous part of this post.

you might also be able to reduce distortion by adjusting the range of your gel. try to make the range of your gel so that your protein is closer to the center of the pH gradient. in your case you will need to run a (very) high pH range gel.

-cuca-

QUOTE (cheeztoast @ Sep 15 2006, 04:32 PM)
Hi all,

I am doing a horizontal Isoelectric focusing gel, see some 6 isoforms with a method.. 8.5-10 pI gradient, 5% gel with urea.. 1.5 hrs (200V) prefocusing.. sample loading and then 1.5 hours focusing (400 V). protein pI ~9.4.

But my bands r constant curves (that's how my boss put it;)).. any ideas to make them straight?

Any reasons for such a thing?

Waiting for quick answers, Thanks!


We┬┤re having the problem that the buffer from the elecrodepaper runs to the side of the gel and therefore the current runs from the opposite electrode through the side of the gel and the bands following the "sideway". In this case take less buffer...

ms-olli

PS:see picture on the left side!!!

-ms-olli-