3 replies to this topic

[Glyoxime]

Hi,

We'be trying to run discontinuous SDS-PAGE gels on a semi-dry flatbed system (Multiphor II from GE). As proposed in the book Electrophoresis in Practice, we used:

Cathodal and anodal Buffer: Laemmli Buffer 5X
Stacking Gel: Tris-HCl 1.25M pH 6.7 / 0.1% SDS
Resolving gel: 12% homogeneous, Tris pH 8.8 like a vertical gel
Power settings: 600V, 50 mA, 22 W, 15 C

It was a complete mess, it took more than 8 h to run. Several bands turned yellow and stopped running down the gel. We tried lowering Tris to 0.125M like a normal vertical gel but the samples were not resolved at all (the MW standard looked OKish).

As everyone, we use the vertical format system for SDS-PAGE, but we were trying to do SDS-PAGE in the Multiphor so that we would have enough practice before doing 2D gels.

Has anyone tried this protocol?

I hope 2D gels are easier...!

[mdfenko]

1.25M tris [final]? that is way too high. the gel formulation should be the same as vertical.

the problem may be how well the running buffer and current are delivered to the gel (we have a multiphor and it says to use filter paper wicks to bring running buffer and current to the gel, i don't know how it says to do it with the multiphor II).

[Glyoxime]

Thanks for the reply.

Yes, it is way too high. We tried it because it says so in the book that came with the Multiphor, but I'm pretty sure it is a typo (Electrophoresis in Practice is full of typos). However, it also did not work very well with an extract with Tris 0.125M (the MW standard run well).

I have also used the paper wicks as it says in the manual.

During the run with Tris 0.125M pH 6.8 (in the stacking gel), when the bromphenol blue entered the resolving gel, two blue bands formed that were 1 cm apart. I think that that caused the loss of resolution. I think there is trouble with the ionic strenght but I cannot find any info online about running SDS-PAGE gels on flatbed systems. There is much more info about SDS gels run on the multiphor when used as the second dimension in 2D gels, but the recipes for the buffers are different.

[mdfenko]

you can try the recipe for the 2nd dimension sds-page (it shouldn't be significantly different from your formulation).

did you use the recommended paper for the wicks? they may have been providing inadequate buffer flow if the wrong grade paper was used.