Ribonuclease Inhibitor – Recombinant, expressed in E. coli

Product No. R1158

Product Description

Ribonuclease Inhibitor, recombinant is derived from E. coli cells expressing a recombinant clone containing portions of the human placental ribonuclease inhibitor. The protein has a molecular mass of ~50 kDa by SDSPAGE.1 Inhibition of ribonuclease activity occurs by the formation of a tight, non-covalent 1:1 complex having a dissociation constant (Ki) of 4 x 10–14 M.1-3

Uses include in vitro inhibition of ribonucleases in procedures such as cDNA synthesis from mRNA, in vitro transcription/ translation reactions,3 ribonuclease protection assays,4 and RT-PCR.5 While this Ribonuclease Inhibitor inhibits RNase A, RNase B, and RNase C, it does not inhibit RNase H, RNase 1, RNase T1, S1 Nuclease, SP6, T7 or T3 RNA Polymerase, AMV or M-MLV Reverse Transcriptase, or Taq Polymerase.

The pH range for inhibition is pH 5.5–9 (highest inhibition at pH 7–8).6 The concentration for use is 250–1000 units/mL.7 Denaturing conditions (i.e., urea or temperatures ≥50 °C) should be avoided as they may cause release of active ribonuclease from the complex. The Ribonuclease Inhibitor may be removed by phenol extraction or inactivated by heating at 65 °C for 10 minutes.

Storage

Storage Temperature –20 °C

Components

This product is supplied in a solution containing 20 mM HEPES-KOH, pH 7.6, with 50 mM KCl, 8 mM DTT, and 50% (v/v) glycerol.

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices. 

Specificity

Activity: 20–40 units/µL.

Unit definition: One unit will cause the inhibition of 50% of the activity of 5 ng of ribonuclease A in a cytidine 2',3'-cyclic monophosphate system.8

Materials

     

 References

  1. Blackburn, P. et al., J. Biol. Chem., 252, 5904 (1977).
  2. Lee, F.S. et al., Biochem., 28, 219 (1988).
  3. Lee, F.S. et al., Biochem., 28, 225 (1989).
  4. Ausubel, F. et al., eds., Short Protocols in Molecular Biology, third edition, John Wiley & Sons, (New York, NY: 1995) p.4-17.
  5. Innis, M.A., and White, T.J., eds., PCR Protocols: A Guide to Methods and Applications, Academic Press, (New York, NY: 1990) p.23 (Catalog Number P8177).
  6. Blackburn, P., and Moore, S., Pancreatic Ribonucleases, in The Enzymes, Vol XV, Part B Academic Press, (New York, NY: 1982).
  7. Farrell, Jr., R.E., RNA Methodologies: A Laboratory Guide for Isolation and Characterization, Academic Press, (New York, NY: 1993) pp. 34-45.
  8. Blackburn, P., J. Biol. Chem., 254, 12484 (1979).
  9. de Martynoff, G. et al., Biochem. Biophys. Res. Commun., 93, 645 (1980).
  10. Scheele, G., and Blackburn, P., Proc. Natl. Acad. Sci. USA, 76, 4898 (1979).

 

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