Ambion® RNase Inhibitor, a recombinant human protein produced in E. coli, is a potent inhibitor of neutral pancreatic RNase A type enzymes. The mode of inhibition is noncompetitive; the inhibitor tightly binds RNases in a 1:1 ratio. Supplied in one tube containing 2,500 U (40 U/ µL). The enzyme has been shown to inactivate RNases present in many tissues and cell types. Addition of the ribonuclease inhibitor (RI) has been shown to be useful whenever the integrity of RNA must be maintained such as in the preparation of cDNA by reverse transcription, in vitro RNA transcription, and in in vitro protein synthesis. RI does not inhibit RNase I, T1, T2, H, U1, U2, or CL3. RI requires a minimum of 1 mM DTT to maintain activity, and has an activity range between pH 5.0 to 8.0 with maximal activity between pH 7.0 and 8.0. Since the mode of inhibition is the formation of a 1:1 complex with RNases, care must be taken to avoid denaturation or oxidation of the ribonuclease inhibitor (e.g. by addition of SDS, urea, etc.). RI should be added to transcription, translation, and cDNA synthesis reactions to give a final concentration of 1 U/ µL. RNase Inhibitor is rigorously tested for contaminating RNase, exonuclease, endonuclease, and protease activity.
One unit is the amount of protein required to inhibit the activity of 5 ng of RNase A by 50%. Unit assay conditions: 100 mM Tris-acetate (pH 6.5), 1 mM EDTA, 1 mM cyclic 2',3'-CMP, and RNase inhibitor. Addition of RNase A initiates the reaction. RNase Inhibitor activity is measured by the inhibition of hydrolysis of cyclic 2', 3'-CMP by RNase A.