Rnase Activity Assay - (Aug/22/2007 )

Hi Everyone

I'm trying to figure out/optimize an Rnase Activity assay from scratch. The purpose is to use RNA + Rnase + Ethdium Bromide and see if I can measure Rnase activity by the decrease in fluorsence (since Etbr only intercalates into unchopped RNA) using a plate spec reader.

But I have no idea how to start, does anyone have any suggestions on what concentration of RNA + Rnase + EtBr I can use?

and which wavelength to read the fluorscence at? and how long I need to incubate the reaction for?


Thanks

Hi There,

I don't think this assay will work, because the fluorescence of the EtBr is not dependent on it's intercalation into the RNA

Nick.

hi there

could you please explain it big more about why EtBr is not dependent on it's intercalation into RNA?
because i thought it's proportional or at least tell you if there is rnase activity since EtBr will bind to uncleaved RNA but not ind to uncleaved RNA

thanks very much

I don't think either this assay would work fine.

RNasa works very quickly plus this way you’ll not able to quantify anything.

Why you want to do this assay??

Do you just want to know what’s the minimum RNasa amount you need to degrade in your sample???
Are you trying to determinate the concentration of the RNasa (unites/ul)??
Could you specify?

i need an essay to be able to monitor rnase activity because the protein i work with is suppose to refold Rnase

check this webpage for an assay technique:

worthington biochemicals