I want to measure the Kd of protein- RNA interaction. What kind of method can be used to get the Kd? I tried ITC but the result was very good. Then I tried EMSA. But as the RNA I use is large, around 511bp, so it is difficult to get through the native gel. now I want to switch it to agarose gel, but I do not know what percentage gel will be suitable and what kind of agarose all be best to run this assay.? what kind of fluorescent reagent could be used to label RNA during the EMSA assay. I really need your suggestion about this experiment. Thank you a lot.
I would try RiboGreen if you need high sensitivity,(https://www.thermofisher.com/order/catalog/product/R11491), but only if you have suitable optics for the wavelengths.
If the RNA is expected to have substantial double-stranded regions, you can probably use any DNA stain, including ethidium bromide.
Hello Ying, I would suggest you to go through this paper which might give you an idea to start with agarose gel for EMSA.
Article Rapid agarose gel electrophoretic mobility shift assay for q...
Hello Ying,
SYBRGold might also be worth a try, although I never used it on RNA or in an agarose gel.
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