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Western gels running super slow!

western gels

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3 replies to this topic

#1 catgirl



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Posted 14 January 2012 - 03:30 PM

I am having some strange problems with running my gels in my new lab. I ran lots of precast gels during my PhD and never saw such issues and I've tried everything I can think of.

Basically I am running standard gels (10-12%), mini format, and they are taking many many hours (6-10+hours), sometimes not finishing ever. At first the buffer was getting very hot and I found out that I was putting the plates in the wrong orientation and didn't have a buffer dam (neither of these were issues with the precast system I used previously). Fixing these 2 issues fixed the heat problem, which was generating so much resistance that the current was maxing out at 400mA.

As part of troubleshooting, my PI suggested that a tech in the lab make a bunch of identical gels and we would troubleshoot in tandem to see what was wrong....when I ran one of his gels, with my samples, it ran perfectly...so troubleshooting over pretty quickly...

Now I made some gels and I'm back to many hours, but no excess heat/current. Of note, I was running 2 gels at the same time, one of which ran about halfway in 6 hours (and the ladder looked fine), and the other, the samples barely made it out of the stacking buffer in 6 hours.

Some of the things I've thought about:
- equipment...I took his gel box the most recent day I tried and still had a problem. I used the same powerbox as I used on the day my gel worked ok.
- the buffers...there is a lab common stock of running buffer, stacking buffer, resolving buffer...others are not having problems so its unlikely the buffer. The tank has plenty of running buffer, and isn't leaking out.
- my samples...don't think source of problem since they are just made by sonicating in PBS with inhibitors.
- number of gels at once - even though the boxes are designed for up to 4 gels at once, it have noticed that my successful run was only on 1 gel at a time. If running 2 simultaneously is a problem....??? any ideas,

Is there something I'm doing wrong in making the gels? That's the only other factor I haven't played around with. The acrylamide solution (we buy Protogel) is relatively recent...only a few months....the buffers are common. SDS is from lab common stock too. I used autoclaved ddH2O. TEMED is a few months old. APS isn't super fresh, maybe a month. My gels appear to have set properly in the normal amount of time...but could something else be going on?

#2 bob1


    Thelymitra pulchella

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Posted 15 January 2012 - 12:07 PM

It sounds like you have a buffer problem - are you sure the buffer you have is not a 5x or 10x stock?

#3 melson17



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Posted 17 January 2012 - 09:37 AM

I would check on the Samples pH or trying a new Sample Buffer. Sometimes I have seen similar results (although never 6+ hours) when any of these solutions were off. The gel just runs slower.

#4 mdfenko


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Posted 19 January 2012 - 09:26 AM

as for running multiple gels, are they identical? if not then the current won't partition equally.

are they both set correctly in the apparatus?
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