RNA EMSA percentage of gel

[jctr]

Hi everyone, i had a question.
I´m doing a RNA-EMSA and I want to use the Mini Protean III system (Bio-Rad) to make the native polyacrylamide gels. The size of my fragments are between 19 and 31 nts. I want to know what is the best percentage of gel than I must use to separate my complexes as well as the ratio of acrylamide/bisacrylamide 29:1, 38:2 or 80:1?

[admin]

Hello JCTR,
i have done similar RNA emsas in the past but with a bit larger fragments. Yours are even a bit smaller than mine and it makes it difficult to see free probe.
You either need to run a gradient gel from low to very high percentage say 15%, or run an overall high percentage gel ie 8-12% and make sure your free probe doesnt come out.

Also when incubating your gel, your very small labeled free probe may diffuse through the gel making it hard to see it. This is a big problem

[jctr]

Thank you for your quick answer. So I already done in largest gels with radioactives probes and I saw the shift bands and the free probe at 6% with ratio of 80:1 but I dont know if these same conditions can work with biotin-labeled RNA and the mini protean III system.

[kevin_a]

Typical DNA EMSA use a 5 or 6% native gel with ~20bp double stranded probe.
Your's is half the mass, so doubling will help, but you'll probably still see diffusion admin pointed out.

You can't just increase the percentage of gel because your proteinrobe complexes are going be very high up on the gel - possibly even look stuck in the well. If you are looking for binding vs no binding it may not matter as much, but if you need to resolve multiple bands you will probably want to tweek with the gradients.

[jctr]

Thank you kevin_a
Other possibility is that I can use agarose gels in TBE buffer for example like done in this paper: Rodgers, J.T. et. al. Analytical Biochemistry [b]277[b], 254-259 (2000). But I think that is more efficient the mini protean III sytem, what do you thinking?

[kevin_a]

In general, you will getter resolution on a polyacrylamide gel. However, agarose allows you separate a larger range of mass... it just depends on what your goal is. Also, since your probes are quite small, they are going to jet to the bottom of the gel quite quickly and you will get more diffusion of the free probe.

The biotinylated EMSA system is great. Furthermore, the detection via chemiluminescence can be as good or better than using 32p. The LightShift EMSA kit from Pierce will give you attamole sensitivity which is as good a 32p. It's a little pricey because it's a kit, but it's similar to doing a Western Blot and I loved using it back in the day. If you decide to go that route, make sure you have good quality TBE buffer and clean transfer materials or you can get rediculous amounts of background when you develop it.

[jctr]

One more time thank you.

I didn´t buy the EMSA shift kit of PIERCE but I did buy the second part that
correspond to the chemiluminescent nucleic acid detection module. I will test it in these days. I will tell you whatever happens with it. See you.

[jctr]

I did a RNA EMSA with the biotin RNA in mini protean II gels in a ratio of 38:2 acrylamide:bisacrylamide and with the chemiluminescent acid nucleic acid detection kit (Pierce). I see perfectly my free probe in the bottom of the nylon membranes but my shift bands are in the upper part and with poor signal. Somebody can give me some suggestions, please.

[kevin_a]

Can you post the image?

[jctr]

Sorry but the web page dont let me load my image I will try in the week ok thanks