no shift for rna-emsa can be caused by:-

- Salt (too much / too little) in the Buffer

- Inappropriate pH

- RNA inappropriately made (the binding area for the protein is not there)

- Binding-protein low abundance due to bad protein preparation (degradation of protein? not enough protein?)

http://www.nature.com/nprot/journal/v2/n8/fig_tab/nprot.2007.249_T5.html

troubleshooting of no shift detected in rna-emsa can be simply explained as stated below:-

1)Disrupted the complex by vortex mixing or heating

solution:-Try running the gel with cold buffer

2)Not enough extract

Solution:-Use more extract

3)Extract degraded

Solution:-Try using protease inhibitors

4)System not optimized

Solution:-Determine effects of additives on the system; for example: KCl, glycerol, NP-40, Mg2+.

https://www.researchgate.net/post/Can_someone_help_me_troubleshoot_my_EMSA_protocol_results

I was going to suggest running the shift with and without the antibody and determining the number of counts in the shifted and supershifted (if there is one) bands. If you get the same number of counts, the antibody doesn't interfere with the binding of the probe to the protein. If you get no shifted or supershifted bands, it means the antibody interferes totally with the binding of the probe to the protein. You could also get other possibilities, like no supershifted but only shifted remaining, which may suggest that the probe-protein interaction is of a higher affinity than the protein-antibody interaction. Of course, any speculation as to relative affinities of the interactions will have to take into account the concentrations of the various components.

A good experiment would be to run the shift with just probe and protein and then add increasing amounts of antibody to the mix to generate a titration curve. If the antibody competes with the probe for binding to the protein then you won't be able to do a supershift. BUT, as long as a negative control antibody doesn't do this, you still have evidence for the probe interacting with the protein you think it is interacting with. In effect, you can still get the information you want whether you can get the supershift experiment to work or not. Good luck!

Problem with my RNA-EMSA is ph of protein..

My Protein is stable in high basic condition more than Ph9 but in this condition generally Protein-RNA interaction disrupts.Finally no shift .

I got Shift may be one or two time by decreasing Ph But whenever I repeat this For good image then problem occurs..

Budak kuantan thank you very much for discussion

Hfq doesnt bind to all types of RNA.

It specifically binds to trans encoded small RNAs. You may not be getting a shift because the protein is not even binding your sRNA. you will get a shift only if the RNA you are targetting is transencoded small RNA, otherwise this just prooves that the small RNA in question may be a cis-encoded sRNA and has greater complementaity with its target and does not need hfq to function.

I need to do a similar experiment, can you please share your protocol for RNA-EMSA?

Hi Shubhangi,

It's been around 4 years I did this. Now I am in completely different field. Please have a look into following paper which we publish. We follow the nature protocol for EMSA & also from kit provider.

'Regulation and RNA-binding properties of Hfq-like RNA chaperones in Bacillus anthracis'

Good luck.

thanks alot for the help. Take care and good luck to you too.