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RiboLock RNase Inhibitor

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RiboLock RNase Inhibitor specifically inhibits the activity of RNases A, B, and C, protecting RNA under a variety of reaction conditions
  
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Thermo Scientific RiboLock RNase Inhibitor inhibits the activity of RNases A (see Figure 1 in Supporting Data), B, and C by binding them in a noncompetitive mode at a 1:1 ratio. It does not inhibit eukaryotic RNases T1, T2, U1, U2, CL3 as well as prokaryotic RNases I and H.

Highlights

  • Performs under a wide range of reaction conditions 
  • Protects RNA from degradation at temperatures up to 55°C (see Figure 2 in Supporting Data)

Note

DTT provided in the Storage Buffer ensures stability during long term storage, but is not necessary for inhibitor activity. Recommended concentration 1 U/µL of a reaction mixture.

  
Storage Condition-20 C
HazardousNo
Concentration40 U/µL
Definition of Activity Unit
  • One unit of the RiboLock RNase Inhibitor inhibits the activity of 5 ng RNase A by 50%.
  • Inhibitor activity is assayed in the following mixture: 100 mM Tris-HCl (pH 7.5), 1.2 mM EDTA, 0.1 mg/mL BSA, 100 ng/mL RNase A, 0.1 mg/mL [3H]-RNA, 50 mg/mL yeast RNA, 8 mM DTT.
InactivationInactivated by heating at 75°C for 10 min. Residual activity detectable after 10 min heating at 70°C.
InhibitionInhibitors: common denaturants (SDS, urea and all oxidizing reagents (p-chloromercuribenzoate, dissolved oxygen, ions in their higher oxidation states) strongly inhibit RiboLock RNase Inhibitor and release the RNase bound.
Molecular Weight49.6 kDa monomer
Quality Control
  • The absence of ribonucleases, endo-, exodeoxyribonucleases and phosphatases confirmed by appropriate quality tests.
  • Functionally tested in RNA and cDNA synthesis.
SourceE.coli cells with a cloned gene encoding mammalian ribonuclease inhibitor.
Storage BufferThe enzyme is supplied in 20 mM HEPES-NaOH (pH 7.5), 50 mM NaCl, 8 mM DTT, 0.03% v/v) ELUGENT Detergent and 50% (v/v) glycerol.
Inhibition of RNase A by RiboLock RNase Inhibitor

Inhibition of RNase A by RiboLock RNase Inhibitor

Inhibition of RNase A by RiboLock RNase Inhibitor

Figure 1 | Inhibition of RNase A by RiboLock RNase Inhibitor.
Total human RNA (1 µg) was incubated at 37°C with 20 U of RiboLock RNase Inhibitor and increasing amounts of RNase A.
M – RiboRuler High Range RNA Ladder
C1 – total human RNA
C2 – total human RNA with RNase A (no RiboLock added)
1-4 – total human RNA with RiboLock and RNase A


Thermostability of RiboLock RNase Inhibitor

Thermostability of RiboLock RNase Inhibitor

Thermostability of RiboLock RNase Inhibitor

Figure 2 | Thermostability of RiboLock RNase Inhibitor.
Total human RNA (1 µg) was incubated with 20 U of RiboLock RNase Inhibitor and 50 pg of RNase A, and incubated at increasing temperatures.
M – RiboRuler High Range RNA Ladder
C1 – total human RNA
C2 – total human RNA with RNase A (no RiboLock added)
1-5 – total human RNA with RiboLock and RNase A


References

  1. D. A. Nielsen, D. J. Shapiro, Preparation of capped RNA transcripts using T7 RNA polymerase.  Nucleic Acids Res. 14, 5936 (1986).
  2. G. Martynoff et al., Synthesis of a full length DNA complementary to thyroglobulin 33S messenger RNA. Biochem. Biophys. Res. Commun. 93, 645-653 (1980).
  3. G. Scheele, P. Blackburn, Role of mammalian RNase inhibitor in cell-free protein synthesis. Proc. Natl. Acad. Sci. USA 76, 1898-1902 (1979).
  4. Van Gelder et al., Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc. Natl. Acad. Sci. USA 87, 1663-1667 (1990).
  5. D. C. Eichler et al., Effect of human placental ribonuclease inhibitor in cell-free ribosomal RNA synthesis. Biochem. Biophys. Res. Commun. 101, 396-403 (1981).
  6. I. J. Polakowski et al., A ribonuclease inhibitor expresses anti-angiogenic properties and leads to reduced tumor growth in mice. Amer. J. Pathol. 143, 507-517 (1993).