11 replies to this topic

[proteinz]

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

[mdfenko]

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?

[rukiye]

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?

Edited by rukiye, 26 March 2009 - 03:52 AM.

[mastermi]

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.

[rukiye]

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.

Attached Thumbnails

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Edited by rukiye, 27 March 2009 - 03:45 AM.

[mastermi]

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.

[Sciurus]

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  

[rukiye]

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.

[Sciurus]

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.

[eldem]

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...

[unity]

APS that is older than one week is fine (in general), but I suggest replacing Aps and Temed anyway with new ones, maybe the problem is with the polymerization of the gel...

weird though

Edited by unity, 30 March 2009 - 11:25 AM.

[rukiye]

Bad gel series coming soon

Series 2

We've got new coomassie blue. We took the previous gels which are dry and contracted and put those gels to Coom.What do you expect? nothing, right? we saw lots of bands clearly.We thought that yes this was the problem, coomassie blue and then casted new gel with new markers,fresh tris buffers and running buffers.Markers are OK. there is no problem in running of markers. At the end, when we put the gel to Coom. again the same result.Thre is no protein on the gel.However, we are sure that there are some as we checked previuos gels with new coom.

for the appearance of gel at the photo, we are thinking that it is due to overloading.the samples are spreading on the gel.What do you think?