Wondering if anyone has experience of working with acid-urea PAGE; I'm trying to run one such gel but encountering problems.

 

Main problem is the samples don't stack.

 

On using a different kind of sample buffer (has glycerol, but not mercaptoethanol) the tracking dye (ethyl green) disappears. It either becomes colourless (reacts?) or instead of entering the gel diffuses out into the buffer (don't know which one).

 

 

Any clues anyone?

What about your protein bands. Do they also disappear?

 

Wondering if anyone has experience of working with acid-urea PAGE; I'm trying to run one such gel but encountering problems.

 

Main problem is the samples don't stack.

 

On using a different kind of sample buffer (has glycerol, but not mercaptoethanol) the tracking dye (ethyl green) disappears. It either becomes colourless (reacts?) or instead of entering the gel diffuses out into the buffer (don't know which one).

 

 

Any clues anyone?

Well, the dye is pH sensitive at pH below 2 (I think) it becomes yellow and often very hard to see. And yes, the next question is whether you manage to stain proteins.

Hi: Thanks for the reply.

 

Yes, I did get protein bands in the case where the sample buffer had glycerol (but overran the gel and probably lost the peptides I'm interested in), but got smears where it didn't.

 

In one experiment I ran both sample buffers in the same gel, and the tracking dye in the one with glycerol disappeared whereas in the other one was visible but did not stack (smeared down the gel).

So it can't be the pH of the gel, otherwise the dye from both buffers should have changed colour.

 

Its hard to find much literature on these gels or their trouble shooting.

 

Oh and I got no bands when I just ran the purified peptides that I intend to use as standards even though I ran the gel for a short time to avoid losing them.

Hi alphas,

 

in fact your results are a bit strange. Probably the polimerization did not occur in a homogeneous way, wich created some pH gradients...

I am sorry but unfortunately I can not help you much more than this...

Have you checked Maniatis book, the "bible" of Molecular Biology, to see if it contains any help?

Yes, they are strange.

 

But thank you anyways.

is there no markers to denote the weight of the target proteins?