I am working on a protein with a probable RNase activity. Both the N & C terminal domains have exhibited the rnase activity .The purified protein domains on incubation with invitro synthesized RNA (approx 300 bases) for 30 mins at 37 degrees followed by etbr based visualization of agarose gel displayed rna degradation. Conataminating rnases were taken care of by carrying out all the steps of purification and elution in buffers made with DEPC treated water. Assay was also carried out with differing concentration of MgCl2 - 0.5-10mM (since some protein require divalent cations for their activity )-some change could be observed. The same assay in presence of different concentrations of EDTA (0.5-20mM), which would probably inhibit the enzyme and show a band shift on the gel could not be observed, infact the protein still showed rnase activity. To check for binding/intercation of the rna with protein, fluorescence based anlysis were carried out by labelling the protein with FITC and then looking for change in signal on addition of RNA, but no such can was observed. Could someone please suggest me a method to quantify the rnase activity and proving the interaction studies, and help me as to what other experiments could be planned?
Hi Hiral,
Perhaps you can start with RNA to detect the RNase activity of your protein. Briefly, 1. Design and synthesize RNA probes; 2. Mix your protein with the reaction system containing RNA probe to obtain the reaction solution; 3. Detect the fluorescence intensity of the reaction solution and identify the RNase activity of your protein according to the fluorescence intensity.
Hope it helps!
Thanks @ Roger.. but what length of RNA would suffice.. also I am unable to get a band shift in EMSA.. The RNA Undergoes degradation in presence of my protein. what can be the solution for this?
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