RNase inhibitor

Molecular biology grade
Cat#RT1061 2000U(40U/µ L)
Store at -20° C
Description: RNasin Ribonuclease Inhibitor is isolated from human placenta. RNasin Ribonuclease Inhibitor inhibits the activity of RNases A, B, C by binding them in a noncompetitive mode at a 1: 1 ratio. It does not inhibit the following RNases: I, T1, T2, H, U1, U2 and CL3.
Molecular Weight: 50 kDa monomer.
Unit Definition: One unit is defined as the amount of RNasin Ribonuclease Inhibitor required to inhibit the activity of 5ng of ribonuclease A by 50%.
Storage Buffer: RNasin Ribonuclease Inhibitor is supplied in 20mM HEPES-KOH (pH 7.6), 50mM KCl, 8mM DTT, 50% (v/v) glycerol.

Applications:
Inhibition of RNA degradation in the following procedures:
- in vitro transcription;
- cDNA synthesis;
- in vitro translation;
- isolation of mammalian cell fractions that contain mRNA-protein complexes;
Separation and identification of specific ribonuclea
Seactivities;
Tumor suppression studies.
Usage Notes: RNasin Ribonuclease Inhibitor is active over a broad pH range but requires a minimum of 1mM dithiothreitol to maintain activity. Concentration gradients may form in frozen products and should be dispersed upon thawing. Mix well prior to use.
Avoid multiple freeze-thaw cycles and exposure to frequent temperature changes.
Quality Control Assays:

RNase Assays: To test for the presence of RNase activity, 1μ G of RNA is incubated with 200 units of native RNasin Ribonuclease Inhibitor for 1 hour at 37° C, and the RNA is then visualized on an ethidium bromide-stained agarose gel to verify the absence of degradation. To test for the presence of latent RNase activity, RNasin Ribonuclease Inhibitor is heat-denatured at 67° C for 15 minutes and the equivalent of 200 units is incubated with 1μ G of RNA for 1 hour at 37° C. The RNA is then visualized on an ethidium bromide-stained agarose gel to verify the absence of degradation.

DNase Assay: To test for DNase activity, 50ng of radiolabeled DNA is incubated with 200 units of RNasin Ribonuclease Inhibitor for 1 hour at 37° C, and the release of radiolabeled nucleotides is monitored by scintillation counting of TCA-soluble material. Minimum passing specification is < 3% release.

Endonuclease Assay: To test for endonuclease activity, 1μ G of supercoiled plasmid DNA is incubated with 200 units of RNasin Ribonuclease Inhibitor for 2 hours at 37° C in Restriction Enzyme Buffer B (6mM Tris-HCl [pH 7.5], 50mM NaCl, 6Mm MgCl2, 1mM DTT). Following incubation, the supercoiled DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible nicking or cutting.
Physical Purity: The purity is >90% as judged by SDS-polyacrylamide gels with Coomassie blue staining.