protein disapeared from gel! - (Feb/03/2009 )

hi all!
i have a unusual problem im trying to load 25ug of protein on a gel. loading on 10ul. i have NEVER had a problem running gels before 5yrs!!! now when i run the gel out the protein just disapears off the gel! usually from the top down??? whats going on? i am running 12% resolving 4% stacking (laemmli). the protein has been running fine before now???
thnks for your time

check and/or replace your electrode buffers.

what do you mean by "protein just disappears off the gel! usually from the top down"?

can you show a picture?

same thing happend to me.I am new in my current lab.I realized that all stock solutions-tris solutions for gel,commasie blue and %20 SDS- stay for a long time on the benches.However, I checked the pH.There is no problem.We prepared fresh running buffer and run the gel using samples with loading buffer which is stored in -20.Also we loaded markers.Nothing seems on the gel.we prepared new sample with SDS loading buffer.same things happend

what is the problem?

It's probably the tris-solution for the gel. Although the pH might still be allright.
Whenever I had this problem it dissappeared after making new tris-solution.

Thank you for your reply mastermi, I tried yesterday but nothing changed. I am not sure but can the problem related with commassie blue?

Also there is some problems with appearance as in the previous gels.I took photo and attached it. Is it normal apperance? I don't think so. the stain should run as a particular band.however, here, there are lower and upper borders.and the stain run dispersed between two borders.

Ok, that looks really strange. I have never seen that before.

Sorry, but I can' help you with this.

Perhaps anybody else had problems like thet before.

Did you stain with Ponceau S to check whether your protein was transfered to the membrane before Coomassie staining?

But your picture looks definitely strange

Sciurus, tahnky you for reply.

I didn't transfer the protein to membrane since I don't see any band on gel after Commassie staining.This is SDS-PAGE gel photo taken after running. this is the third gel with same appearance. we ordered new commasie blue but I don't think the problem is related to cammassie blue.

I am stuck in and need help.Please anybody:((( otherwise I cannot do further experiments.

Oh okay, my mistake then.

Maybe you should run a gel again after exchanging every chemical you used for making it; buffer, acrylamide, temed, etc. If your problem persist you can at least exclude that some of your chemicals were degraded or something.

Hi....
I don t know if i can help but please chech your 10%APsif it s more than a week don t use it.....I have done it once by mistake and i had strange loading buffer running....
Good luck...