protein disapeared from gel!
Posted 03 February 2009 - 05:57 AM
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
Posted 03 February 2009 - 08:38 AM
what do you mean by "protein just disappears off the gel! usually from the top down"?
can you show a picture?
genius does what it must
i do what i get paid to do
Posted 26 March 2009 - 03:51 AM
what is the problem?
Edited by rukiye, 26 March 2009 - 03:52 AM.
Posted 26 March 2009 - 04:45 AM
Whenever I had this problem it dissappeared after making new tris-solution.
Posted 27 March 2009 - 03:44 AM
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.
Edited by rukiye, 27 March 2009 - 03:45 AM.
Posted 28 March 2009 - 10:54 AM
Sorry, but I can' help you with this.
Perhaps anybody else had problems like thet before.
Posted 28 March 2009 - 01:01 PM
But your picture looks definitely strange
Posted 30 March 2009 - 07:49 AM
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.
Posted 30 March 2009 - 08:00 AM
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.
Posted 30 March 2009 - 08:30 AM
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....
Posted 30 March 2009 - 11:23 AM
Edited by unity, 30 March 2009 - 11:25 AM.
Posted 02 April 2009 - 06:32 AM
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?