Help!! SDS page problem - (Apr/28/2009 )

Hi,

Recently I have been getting wide cube-like bands on the lower portion of my gels (the top of the gel is fine). I have been running the same 5kDa protein on 15% gels for months without an issue (180V 50mins) but a few weeks ago it started running wider. Over the past few weeks I have tried so many things to correct it, making the solutions fresh, increasing the %age to 16.5 and 17.5, using different acrylamide, running at lower voltage, decreasing the sample volume and increasing the size of stacking gel. I know the samples (all aliquoted out and frozen - they have kept much longer in the past) are all fine. They contain 720ng protein of interest and no added salt. I feel like I'm going crazy. Please help

Jenny

did you change lot# of the stock chemicals before the problem started?

how old is your sds?

did you polymerize the gel for the same amount of time as before the problem occurred?

is the temed starting to turn yellow (or is it already yellow)?

is your persulfate stock powder dry and free flowing?

can you post a picture so that we can see the problem?

I've uploaded 2 gels, each with repeat samples from the same experiment. The better one was an old gel that I had actually stored @ 4C for a week before i used it. The other was made the day of the experiment and ran the following day. Since this problem I suppose I have been in gel making overload and have used the gels perhaps an hour following them being set to find out if the thing I changed worked. As you can see the markers have the same problem towards the bottom of the gel so it isn't my samples. I don't believe the lot # changed with any of the chemicals, we buy in bulk and make a lot of gels as a group. The APS powder is fine and stock solution has been made up fresh several times since. The temed is perhaps slightly off clear, perhaps I should order some more. I have not changed the SDS solution. Other peoples gels look fine using these solutions, although they are looking for larger proteins. I have noticed that while the gels are running the blue buffer colour seperates instead of condensing. Does this help. If you have a solution I would be so greatful.

Jenny

For me this looks like your gels are leaking. That could be dut to the gels themselves or the gel chamber. If your glass plates or spacers are just alittle uneven, or the gels aren't fitted tightly in the chamber, the running buffer would leak out of the sides of your gel.
So maybe try it another time with the glass plates, spacer and chamber of one of your colleagues who doesn't have this problem.

Hmmmm...gel leaking.....I doubt if that is the reason. I have seen similar gels in my case and they never leaked. Besides we have a common stock of plates, spacer and all and this particular protein that I am talkign abt ran like this in all cases. Like mdfenko I also think that it has more to with the reagents than anything else. But It would be interesting to know

Best,
TC

mastermi on May 1 2009, 02:34 PM said: