Problem with EMSA - Undefined bands

[amazonic9]

Hi everybody,

I'm trying to optimize EMSA for NFkB at the laboratory I am working at.
When I perform the EMSA, I can see the shift and even a supershift with some of the NFkB subunits, but the "image" I get isn't what I expect. The thing is that instead of having well defined bands, I have blured spots. I'd like to show you one of my EMSAs but as I have only 2 messages posted I can't do that.

Can somebody help me? I've changed different things trying to improve it (Buffers, timing, etc.) but with no result. I use Novex 6% retardation gels.

Any contribution will be wellcome.

Thanks.

[amazonic9]

No ideas??

[kmunson779]

It's hard to make suggestions when we have no idea what your current protocol is like. Are you pre-running your gel? What buffers have you tried? What kinds of samples are you running (whole cell extracts/ purified protein positive controls)? Can you reduce the volume of sample you're loading? A picture would really help; can you post it on a third party site and give us the url modified with some extra bits (like spell out 'dot', add spaces, etc.) to workaround the link restrictions?

[amazonic9]

Thanks for answering.

I pre-run the gel although in the instructions it does say that it's not necessary to do it.
I usually run nuclear extracts, and sometimes total extracts, and I have the same image with both of them.
I use 3ug of protein to do the emsas.

Here is an image for you to have an idea: h t t p : / / mail.google[dot]com/mail/?ui=2&ik=e1c4f7162a&view=att&th=116a503bbecbc305&a ttid=0.1

[kmunson779]

Your link isn't working for me... it looks like an email in your Gmail account, and I'm not going to be able to see it. Try uploading to [Only registered users see links. ], [Only registered users see links. ] or [Only registered users see links. ]?

[amazonic9]

Now?

h t t p : / / w w w .flickr[dot]com/photo_zoom.gne?id=2114993118&size=o

[kmunson779]

Closer! I managed to figure out where the picture was from your link. [Only registered users see links. ] for anyone else paying attention.

It looks okay to me; your bands are a little taller than I'd like to see but not bad. My first suggestion is to try a shorter exposure, you've got plenty of signal and bringing down the background could help.

Second, could you reduce the volume you load into your wells? There isn't good stacking action in EMSA gels, so the height of the sample you load will tend to be the height of your band. If you can load a smaller volume, the height of the sample will be shorter and your band should be too.

You have a lot of unbound probe. Try titrating down the amount of probe you add (try a 2- or 4- fold dilution series) while keeping the protein amount the same. Along similar lines, running the gel longer may help clear some of the unbound probe signal out of the area where you see your shift.

The experimental results look pretty clear to me, now it's just getting the picture prettier to show it off!

[admin]

Hello there are several things with EMSA to consider, I have done many (hope I never do them again! )

To have sharp EMSA bands you need:

• TBE works best for EMSAs
• cool gel running temperature (I would run the gels in a 4C cold room)
• Percentage of gel and how it was made
• Are you using non-denaturing or denaturing (SDS and dmso) conditions?

Non-denturing gels are by standard less sharp due to the absence of sds and dmso. Try optimizing your running conditions and gel making, and you should get a bit sharper bands.

Supershifts require very exact conditions and the conditions (salt, buffers) may affect the gel running (Band sharpness).


Pretty decent emsa gels though especially with supershifts good for you!

[amazonic9]

Thank you very much for your answers.
I'll try to concentrate my binding buffer (I have it at 2x) to reduce the volume I put. Usually I load 10 ul in each well.
I'll do the titration of the probe too. Thanks.

I use TBE 0.5X in my EMSAs, and I run them in cold temperature (I don't know exacly what temperature, because as I can't put the electrophoresis in a cold room, what I do is to put next to the tank two recipients with blue ice, which mantains it cold.)

The gels are 6% retardation gels from Novex. I think they are non-denaturing.

Thanks you two for your suggestions. They are realy wellcome. I'll try to improve my conditions and hope I'll get a better image. If you think of anything else, again it will be really wellcome.

Thanks.

[admin]

Hello Amazonic,
yes please let us know what happens next time you run it

Good luck!