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| Product Name | Ribonuclease Inhibitor (RNasin), Recombinant |
| Catalog No. | NATR-HMM-0143 |
| Description | A recombinant human placental ribonuclease inhibitor protein expressed and purified from E. coli. This 50 kDa protein specifically binds to and non-competitively inhibits pancreatic-type ribonucleases including RNase A, RNase B, and RNase C, providing essential RNA protection during enzymatic reactions. |
| Intended Use | Protection of RNA from degradation by contaminating RNases during reverse transcription, in vitro transcription, in vitro translation, RNA labeling, and other enzymatic procedures where RNA is the substrate or product. |
| Principle / Technology | The recombinant ribonuclease inhibitor binds to pancreatic-type RNases in a 1:1 stoichiometric complex with an extraordinarily high affinity (Ki ~4 × 10^-14 M for RNase A). Binding occurs through extensive protein-protein interactions that sterically block the RNase active site. The inhibitor is specific for neutral pH-active RNases and does not inhibit RNase H, RNase T1, S1 nuclease, or other ribonucleases with markedly different catalytic mechanisms. |
| Detection Method | RNA integrity verified by denaturing agarose or polyacrylamide gel electrophoresis before and after enzymatic incubation; Bioanalyzer RIN analysis for quantitative RNA quality assessment; RT-qPCR amplification of reference gene as functional endpoint for RNA preservation; in vitro transcription yield as functional measure of RNA protection |
| Sample Type | All enzymatic reactions containing RNA as substrate or product: first-strand cDNA synthesis, one-step RT-PCR and RT-qPCR, in vitro transcription, RNA labeling, cell-free translation with RNA templates, RNA ligation, and RNA 5ʹ-end modification |
| Performance Range / Specifications | Activity: ≥40 U/μL (one unit inhibits 5 ng of RNase A by 50% in a standard cCMP hydrolysis assay); recommended usage: 1 U/μL final concentration (0.5–1 μL per 20 μL reaction); active pH range: 5.0–8.0; optimal pH: 7.0–7.5; reducing agent requirement: 1 mM DTT minimum for maximal activity |
| Sensitivity / LOD | One unit of inhibitor protects 1 μg of RNA from degradation by 5 ng RNase A for >30 minutes at 37°C; 20 U in a 20 μL RT reaction protects RNA from RNase contamination up to 50 pg throughout standard 1-hour synthesis |
| Specificity | Specific inhibitor of neutral, pancreatic-type RNases (RNase A, B, C family); does not inhibit RNase H, RNase T1, S1 nuclease, or T2-family RNases; no detectable DNase, non-specific endonuclease, or protease activity in recombinant preparation |
| Reaction Conditions / Protocol | Add 0.5–1 μL of recombinant ribonuclease inhibitor (20–40 U) per 20 μL reaction after RNA addition; include in all reactions where RNA is present; maintain reducing environment with ≥1 mM DTT (supplied in most RT and transcription buffers); inhibitor requires DTT for stability and activity; add RNAse inhibitor before adding RNA to the reaction mixture |
| Components / Formulation | Recombinant human placental ribonuclease inhibitor (50 kDa, E. coli-expressed, ≥40 U/μL) in storage buffer (20 mM HEPES-KOH pH 7.6, 50 mM KCl, 8 mM DTT, 50% glycerol, 0.5% (v/v) NP-40) |
| Storage Conditions | –20°C for long-term storage; stable at 2–8°C for up to 1 month; avoid temperatures >25°C (thermal denaturation begins above 37°C); do not freeze-thaw more than 10 times; aliquot to ensure fresh inhibitor for each experiment |
| Shelf Life | 24 months at –20°C from date of manufacture |
| Package Specifications | 2,500 U, 10,000 U, and 50,000 U packaging; sufficient for 125, 500, and 2,500 standard reactions respectively |
| Product Form | Liquid enzyme in glycerol-containing storage buffer (50% glycerol prevents freezing at –20°C) |
| Quality Control | Each lot tested: specific activity ≥40 U/μL; RNase A inhibition verified by agarose gel assay (1 μg RNA protected from 5 ng RNase A for 30 minutes at 37°C with 20 U inhibitor); DNase-free (no degradation of 1 μg DNA in 16 hours); no non-specific RNA binding; functional in reverse transcription (cDNA yield comparable to freshly opened enzyme standard); endotoxin <1 EU/mg protein |
| Key Features | Recombinant production from E. coli eliminates potential contamination from human pathogens; 1:1 stoichiometric binding provides reliable, quantifiable RNase protection; essential for sensitive single-cell RNA-seq and low-input RNA experiments; broad pH tolerance (5.0–8.0) accommodates diverse enzymatic reaction conditions; DTT requirement compatible with all standard reverse transcriptase and RNA polymerase buffers |
For research use only, not for clinical use.
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