This has really been quite frustrating. Every time I load my samples (20 to 45 ug per well), I see lines form in the resolving gel and they interrupt the run. The proteins just accumulate on that line and seem to disappear (I can tell based on the ladder). If I simply run the gel for a longer time and wait for the line to run through the gel, this doesn't happen. Bubbles form under the line and the gel is just ruined and useless. I normally prepare my samples in Liammeli buffer with B-ME by heating for 5 min at 85 degrees C. I have never had this problem before in my previous labs but I am encountering it now in a new lab. Does anyone know why these lines are forming and how I can address them?? I have tried everything from precast gels, making my own Liammeli dye or commercial ones, remaking ALL the reagents fresh, using low or high voltage to run the gels, and even using cold packets to lower the heat in the system. I don't know what else to try! What is going on??! Help!
Hi Erome;
Did you try to use another electro-kit? I mean, if the buffers and the samples are well done... it´s logic to think that maybe there is a problem with your hardware. I don´t know ...
Maybe if you can use another western blot set ( another energy source, another gel support...)
hope it helps!
Hi Oscar, I have already tried that. It was the first thing I tried, actually. I am as stumped as you are...I don't know what the problem could be...
Good question.
Realy it's not easy to fix the problem, and I faced a similar problem on SDS_PAGE.
Try to prepare the acrylamide gel again, and try to mix the gel well before pouring in the apparatus.
I think there is no problem in the sample buffer, because the most troubleshoot come from it.
Good luck
Sorry, I think there is no problem in the sample buffer although most troubleshoot of SDS_PAGE come from it
Thanks everyone. I'll keep working on resolving this (pun intended).
Erome Daniel Hankore Oscar Recasens Khaleel Z. K. Al-Alo
Can you tell me what is the problem in here SDS-Gel ? this is OMVs sample . why I did not get so many protein in gel ? why I get only one ?
Nazmul Islam
I think You have to increase the amount of the loaded protein sample, and change the concentration of the gel.
Better to try the two probabilities.
Then till me if it's okay or not.
Hi, hope you have all your answers! If not, I would love to add some.
Is anyone else having the same issue in your lab? I suspect the trouble you are FACING might come from THREE major sources--- power supply, gel (glass+gel itself), Running buffer.
You may try preparing your samples in 5X Sample buffer with 5% 2ME denatured for 10 min at 100 degree C.
Load 30ug test sample in 10% gel (suspend the gel carefully)--you may use commercial gel as well. If you have easy access to commercial gel, I would suggest you to try that first. So you can be 100% sure of your gel quality..... If you still face that problem , maybe change your apparatus and buffers ...
Better, check first if anybody else in your Lab having this same issue.
BTW, before preparing gel do you clean your glass by 95% EtOH using KIMWIPES? If not, pls do this..
Good luck.
Hi everyone, I wanted to let you know this issue has been resolved. I think there may have been something wrong with the quality of the chemicals I was using to make running buffer because using commercial running buffer was the answer.
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