EMSA Problem - (Feb/09/2007 )

Hallo,
I am now performing EMSA using a LightShift chemiluminescent EMSA kit (Pierce).
However, i can't get any bands, sometimes, there are just some spots on the top, that is the same even with the positive control. Can someone here give me suggestions and help? Here is my protocol:

After binding reactions, the sample was ran in a 6% polyacrylamide gel at 150v for 1 hour, following a semi-dry tranfer (30v 45min.)and cross-link with a transilluminator with 312 nm bulb for 10 minutes. Detetion was perfomed just described in the kit, ?

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Are you saying that you don't even see the free probe band? If this is the case it could be the transfer stage that is the problem. Either no transfer is occuring (try stainign the gel e.g. with coomassie blue to check that transfer is complete) or the transfer satge is too long. Is your loading dye transfering to the membrane? I'm affraid I'm not familair with your blotting technique but using a wet transfer I found that the gel blotted very quickly. What type of membrane are you using I know that some do not work for EMSAs?

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Yes, there is even no free probe band. I used the Biodyne B positively chared nylon membrane from PIERCE, after electrophorse the loading dye already migrated out of the gel, therefore i can not check the transfer efficiency. How about using a protein marker during electrophorse? Is it possible to perform a coomassie blue before cross-link?

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What's the size of your oligo? If it's not very long it might have already migrated out of the gel. I use an oligo that is 25 bp and it migrates in front of the dye so I stop the electrophoresis before the dye has reached the bottom of the gel.
When you say that you can't even detect the positive control, do you mean the EBNA one in the kit or your free probe?

I'm also using biotin labeling and semi dry transfer (20V, 90 min) to a positive nylon membrane (Amersham) and that part of the EMSA is working... I'm struggling with getting a specific shift...

If you use a prestained protein marker you won't have to do a coomassie blue staining.

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The oligo i used is ca. 30bp. Yesterday I tried only the positive control, i can detect the EBNA band this time, however it is not a "real" band, but a large speck. I am wondering if the voltage (150v) i am using is too high.
At what voltage did you run the electrophoresis? and how did you perform the cross-link?

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I suspect your stuff isn't entering the gel. I would think your problem lies there. I wouldn't be as worried about the voltage, as about the composition of your binding buffer

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I used a 5% acrylamide gel, what problems can be if the samples do not enter the gel?

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your binding buffer components are very important here

if your proteins are making a big gob in the binding reaction, they wont' always enter the gel well

depends on the protein, too...I would fiddle with the buffer composition. I read a reference a few years ago when I was starting to do EMSAs (don't remember it, you'll have to dig yourself, sorry!) that said an author actually had to add BSA to the electrophoresis buffer to get everything to enter the gel properly

however, this only addresses the issue of why you're not seeing your positive control

it seems like your transfer could be bad as well; or at least not optimal. your semi-dry v/time seems sort of high for something tiny like oligos

because both the little things and the big things are not showing up on your blot properly, there may be an underlying larger issue. would you post a picture? have you checked everything, like remaking your buffers and double-checking their pH? do you follow Pierce's protocol exactly, or do you fiddle with it? has your semi-dry apparatus been used successfully lately? have you performed an s/d transfer successfully yourself?

anyways, don't know if I've been helpful, but trying to help you think of things?

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