Hi all,

I have been a running a 6% gel for my EMSAs, and have been seeing some of my protein stuck in the wells. This has recently become more accentuated, and my last gel had no bands, as all the protein was stuck in the wells! I am using HeLa extract, as well as nuclear extract from rat brain tissue as my protein source. I also add an oligo that is my target sequence. I use the binding buffer provided by Promega (20%glycerol, 5mM MgCl2, 2.5mM EDTA, 2.5 mM DTT, 250mM NaCl, 50mM Tris-HCl, 0.25mg/ml poly dIdC).

For my binding reaction, I add 5ul of the binding buffer, 2.5ul of HeLa or 10ug of protein from nuclear extract, 2ul ultrapure H2O and 1.5ul of labelled probe. I then let this incubate for 10 minutes at Room Temp, then add 2ul of loading buffer.

Please let me know what you all experts think is the problem.

Thanks in advance,

Hasan

Hi! I was wondering if you found the problem with your EMSA's. I've been working with this technique for a year now, and at the beginning I had this problem, then it somehow resolved.
But now I have the same problem again! I've tried changing all possible factors: acrylamide, buffer, nuclear extracts, probe, temperature... I don't have a clue why this happens sometimes, and so I thought of writing you, if you've solved this I'd very much appreciate a lot if you could reply.
Thank you so much in advance,

Olivia

It sounds like a problem with gel. I normally wash my wells out a few times with the EMSA running buffer (0.5X TBE) before I run it to get rid of any non-polymerised acrylamide which could stick to the probe and hold it in the wells. I don't normally pre-run my gels but maybe that would also help. If nothing is running into the gel has your acrylamide changed i.e. are you running higher % gels or is your electrophoresis power pack not working properly or your TBE is not right (is the current lower than it should be, has the EDTA come out of solution).

All the best,
Ceri

do you pre-run the gel before to load the sample?
I don't know exactly what it is for. It was a trick told to me, and I never tried without, and it was working.

You just have the gel, gel tank and buffer and run it for 30mins/1hr at the right voltage so you get a constant current. Then when you samples are ready, stop the gel, load them and restart the gel. It's supposed to give better results. I've not really been doing it as the results I get seem to be fine and it's extra hassle to dismantle the gel tank.

All the best,
Ceri

Try adding 0.1% final w/v detergent (say Triton X-100) to you binding buffer. This can sometimes help.

Have you glass plate been scratched? You might try to siliconise them or replace them if they're damaged.

Make sure your wells are properly flushed out before loading. What's this loading buffer? Don't see why you need it. The binding buffer seems quite adequate to me for weighing down your samples.

Finally, reduce the amount of crosslinking in your gel by using a more dilute acrylamide mix. Try 37.5:1 rather than the standard 19:1.

Sometimes EDTA can cause hanging lanes. It's not entirely necessary to be in the buffer. Try making your own buffer without it but with detergent and more poly(dIdC)-poly(dIdC).

I always prerun my gels before loading the samples. I do the prerun at 150V for 30 min and the "real" run at 200V for 2h. You could also try to increase your glycerol concentration. Are your samples very viscous? Perhaps you could make a benzoenase digestion to get rid of the nucleicacids in the protein samples, then they are more liquid in running better in the gel. Or try to reduce your amount of acrylamid.

Hi,

I also have this "well" problem, although I have tried almost all suggestions!
I do pre-run the gel and flush the wells before loading the samples.
Is it possible that some oligonicleotide probes are more prone to multimer formation and thereby precipitate?

If somebody has solved this problem or has an additional suggestion, please let me know!

Thanks in advance,

Ines

Hi Ines,

did you solve your well problem?
Because we have the same problem, we tried a lot of suggestions but nothing!
We are almost hopeless......

Thanks in advance.
Aquila's lab.

I am wondering whether you add the loading dye. I tried to reduce the amount of probe and didn't add the loading dye. It worked well for me.

zoux