Available Options:
| SKU | Unit | ||
|---|---|---|---|
 | RT35L-B | Bulk | Ask for an offer |
| RT35L-010 | 10 000 U ( 40U/μl ) | Ask for an offer |
The RIBOPROTECT Hu RNase Inhibitor Lyo-Ready is a 50 kDa recombinant human placental protein expressed in Escherichia coli, in a special formulation (without glycerol). It inhibits ribonuclease (RNase) activity of common eukaryotic enzymes such as RNase A, RNase B, RNase C by non-covalent binding in a 1:1 ratio. RIBOPROTECT Hu is intended for use in applications where the presence of RNases may cause a hazard to RNA quality and experiment results, e.g. in RNA isolation, cDNA synthesis, RT-PCR, RT-qPCR, RT-LAMP, in vitro transcription and translation, or RNase-free monoclonal antibody preparation. RIBOPROTECT Hu shows no activity towards RNase 1, RNase T1, RNase T2, S1 nuclease, and RNase H. It is compatible with DNA Polymerases and AMV or M-MuLV Reverse Transcriptases. Formulation of RIBOPROTECT Hu RNase Inhibitor Lyo-Ready enables its usage directly in the lyophilization process.
Features and advantages
- Formulation without glycerol for efficient freeze-drying
- High functionality after lyophilization and reconstitution
- Stable at 37°C for at least 4 weeks with no loss of activity
- Fully stable for at least 6 hours in 50°C
- Completely inhibits RNase A, RNase B, and RNaseC activity
- Possibility of single-reaction or in-bulk lyophilization process
- No influence on the polymerase or reverse transcriptase activity
- Free of DNase and RNase activity
- Active in diverse reaction conditions and in various buffers
- Active over a broad range of pH and DTT
- High concentration formulation available (100 kU/ml)
Applications
- cDNA synthesis, RT-PCR, RT-qPCR, RT-LAMP
- Optimal RNA protection in lyophilized diagnostic kits
- RNA isolation and purification
- in vitro transcription and translation
- RNase-free monoclonal antibody preparation
Unit definition
One unit is defined as the amount of enzyme required to inhibit the activity of 5 ng RNase A by 50%.
Lyophilization with different additives
A number of different additives, most commonly used in lyophilization, have been added to the RIBOPROTECT Hu Lyo-ready RNase Inhibitor (Table 1). After lyophilization, the samples were dissolved in DEPC water and analyzed for functionality.
5 U of RNase Inhibitor was incubated with 1.5 ng of RNase A and 1 µg of eukaryotic RNA from trout liver for 15 minutes at 37˚C.
Results were visualized in non-denaturing agarose gel (1%). Protected RNA is represented by two clear bands.
RIBOPROTECT Hu RNase Inhibitor remains fully active after the freeze-drying process with or without various additives.
Stability of RIBOPROTECT Hu LYO-READY
RIBOPROTECT Hu Lyo-ready RNase Inhibitor was formulated without glycerol at 40 U/µl and 160 U/µl concentrations and stored for 4 weeks at 37˚C. After 4 weeks, the samples were reconstituted and tested for RNase Inhibitor functional activity.
5 U of RNase Inhibitor was incubated with 1.5 ng of RNase A and 1 µg of eukaryotic RNA from trout liver for 15 minutes at 37˚C.
Results were visualized in non-denaturing agarose gel (1%). Protected RNA is represented by two clear bands.
K – Sample without RNase A, without RNase Inhibitor; 1, 1’ – RIBOPROTECT Hu Lyo-ready RNase Inhibitor, 40 U/µl; 2, 2’ – RIBOPROTECT Hu Lyo-ready RNase Inhibitor, 160 U/µl
RIBOPROTECT Hu Lyo-Ready remains fully active after storage for at least 4 weeks at 37˚C.
Temperature stability of RIBOPROTECT Hu RNase Inhibitor Lyo–ready
RIBOPROTECT Hu Lyo-ready RNase Inhibitor (two batches – A, B) and RIBOPROTECT Hu RNase Inhibitor (one batch – C) were incubated at 50˚C for 6 hours in order to test the stability.
40 U of both versions of RNase Inhibitor was incubated with 5 ng of RNase A and 1 mg of eukaryotic RNA from trout liver for 15 minutes at 37˚C.
Results were visualized in non-denaturing agarose gel (1%). Unaffected RNA should be represented by two clear bands (K-). Results: No discrepancies between RIBOPROTECT Hu RNase Inhibitors – both remain fully functional after at least 6 hours of incubation at 50˚C.
RIBOPROTECT Hu Lyo-ready remains fully active after at least 6 hours of incubation at 50˚C.
APPLICATION of RIBOPROTECT Hu RNase Inhibitor Lyo–ready in RT-LAMP viral detection tests
Reaction mixture containing RIBOPROTECT Hu Lyo-ready RNase Inhibitor, Reverse Transcriptase and Bst Polymerase was lyophilized in the conditions as follows:
Manual mode, without heating the shelves, pump heating 20 minutes, basic drying: pressure 0.01 mbar, time: 15 h
After the lyophilization process, the obtained reaction mixes were subjected to quality control with the same templates (3 serial dilutions of human RNA) as for the control of the reaction mixture before lyophilization.
Results: The conducted studies showed that the freeze-dried reaction mix with RIBOPROTECT Hu Lyo-ready RNase Inhibitor, Reverse Transcriptase and Bst Polymerase retain full activity and functionality. Ct values indicate high efficiency and performance in SARS-CoV-2 detection.
RIBOPROTECT Hu Lyo-ready is succesfully used in the RT-LAMP reaction.
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Quality control
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Functional activity test
RIBOPROTECT Hu Lyo-ready RNase Inhibitor activity is assayed in RNase A inhibition in a reaction containing 1 µg of RNA and various amounts of inhibitor for 15 minutes at 37˚C. Results are visualized by agarose gel electrophoresis.
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DNase (endonucleases, exonucleases), and RNase activity
Free of detectable nuclease activities as judged by gel electrophoresis following incubation of 200 U of RIBOPROTECT Hu Lyo-ready RNase Inhibitor with 1 µg of DNA or RNA in a reaction for 1-2 h at 37˚C.
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Physical Purity
The purity is >90% as evaluated by SDS-PAGE electrophoresis.
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