RNase Inhibitor Instructions

RNase Inhibitor Instructions

Composition

Storage

It can be stored for up to two years at -20℃.

Introduction

RNase Inhibitor (RNasin) is a recombinant RNase inhibitor expressed and purified in E. coli in soluble form, which has the same application effect as a specific ribonuclease inhibitor present in the human placenta and is essentially a protein with a molecular weight of 51,000 Da and an isoelectric pH of 4.7.

RNase Inhibitor can specifically bind to RNase A, B, and C with non-covalent bonds to form a 1:1 complex, thereby inactivating RNase, and has a broad spectrum of RNase inhibitory activity. It is active at buffer 0-0.5 M NaCl, pH 5-8, and is most active at pH 7.8. RNase Inhibitor can protect the integrity of mRNA, which is beneficial to improve the efficiency of transcription and translation, while avoiding the possible effects of using organic compound inhibitors.

RNase Inhibitor has been tested by RT-PCR and RT-qPCR and is compatible with various reverse transcriptase and DNA polymerase. Compared with human RNase inhibitors, recombinant RNase inhibitors do not contain two cysteines, resulting in higher antioxidant activity and better suitability for experiments sensitive to high DTT (e.g., qPCR).

Storage Buffer

20 mM HEPES-KOH (pH 7.5), 50 mM KCl, 5 mM DTT, 50% Glycerol

Unit Definition

The amount of enzyme required to inhibit 50% of 5 ng RNaseA activity is defined as one unit of activity (U). (Inhibitory activity is determined by inhibiting RNaseA's ability to hydrolyze Cyclic2',3'-CMP)

Purity

1.      The electrophoretic bands of 300 units of RNase Inhibitor and 1 μg of super helical pBR322 DNA were not changed after reaction at 37℃ for 1 h.

2.      The reaction between 100 units RNase Inhibitor and 1 μg 16S, 23S rRNA at 37℃ for 1 h showed no change in the electrophoretic bands of RNA.

3.      SDS-PAGE: Single band was observed at molecular weight 50 KDa.

Recommended Concentration

1.      cDNA synthesis reaction：0.5 U/μl of a reaction mixture.

2.      In vitro translation：1 U/μl of a reaction mixture.

3.      In vitro cell-free transcription：20 U/μl of a reaction mixture.

4.      In vitro transcription of SP6 or T7 RNA polymerase：1 U/μl of a reaction mixture.

5.      Multiple ribosome separation：1 U/μl of a reaction mixture.