RNase A, CAS No.9001-99-4

CAS: 9001-99-4 Cat. No.: R1522663 Número CE: 232-646-6 PubChem CID: 9369

GRAU & PUREZA Bioactive, ActiBioPure™, DNase free, Native, High Performance, Suitable for molecular biology, EnzymoPure™, ≥85%(SDS-PAGE), from Bovine Pancreas; ≥80 Kunitz U/mg enzyme powder

Accession #

P61823

Bioactivity

≥80 Kunitz U/mg enzyme powder

Visão geral

In addition to polymerase activity, this DNA polymerase exhibits 3'-5' and 5'-3' exonuclease activity. Ribonuclease A, commonly abbreviated as RNase A, is an endoribonuclease containing four disulfide bonds with a molecular weight of approximately 13.7 kDa. It specifically targets the 3' side of pyrimidine residues in RNA and cleaves the phosphodiester bond with the adjacent nucleotide. The end products are pyrimidine 3'-phosphates and oligonucleotides terminating with pyrimidine 3'-phosphates. RNase A exhibits the highest cleavage activity toward single-stranded RNA and displays differential cleavage behaviors under varying reaction conditions. At low salt concentrations (0–100 mM NaCl), it cleaves single-stranded RNA, double-stranded RNA, and the RNA strand within RNA‑DNA hybrids. At high salt concentrations (≥0.3 M), RNase A specifically cleaves only single-stranded RNA.

Applications

1. Removal of RNA during plasmid and genomic DNA extraction.
2. Elimination of RNA contaminants from recombinant protein preparations.
3. Ribonuclease protection assay.
4. RNA sequence analysis.

Quality Control

Protein Purity

SDS-PAGE analysis shows the protein purity is no less than 85%.

DNase Activity

10 μg of RNase A was incubated with 15 ng double-stranded DNA fragments in a 20 μL reaction system at 37 °C for 16 h. Agarose gel electrophoresis showed no degradation of the dsDNA substrate.

Functional Activity

5 ng of RNase A was incubated with 1 μg RNA in a 10 μL reaction system at 37 °C for 1 h. Agarose gel electrophoresis confirmed complete degradation of RNA.

Instructions for Use

1. This product is DNase-free and ready for use after dissolution. The recommended working concentration is 1–100 μg/mL. The suggested storage buffer contains 50 mM Tris-HCl (pH 7.4) and 50% glycerol.
2. RNase A cannot be heat-inactivated. It is recommended to remove the enzyme completely by spin column purification or phenol-chloroform extraction.

Especificações

Product Name

RNase A, CAS No.9001-99-4

Sinónimos

Ribonuclease A | pancreatic ribonuclease | pancreatic RNase | ribonuclease I | RNase | RNase A | RNase I | RNAsea | RNASE1 | RAC2 | RNS1

Grau

ActiBioPure™, Bioactive, DNase free, High Performance, Native, Suitable for molecular biology, EnzymoPure™

Especificações e pureza

Bioactive,ActiBioPure™,DNase free,Native,High Performance,Suitable for molecular biology,EnzymoPure™,≥85%(SDS-PAGE),from Bovine Pancreas; ≥80 Kunitz U/mg enzyme powder

Mecanismos bioquímicos e fisiológicos

Endonuclease that catalyzes the cleavage of RNA on the 3' side of pyrimidine nucleotides. Acts on single-stranded and double-stranded RNA.

Bioatividade

≥80 Kunitz U/mg enzyme powder

Número de acesso

P61823

Peso molecular previsto

13.7 kDa

CAS

9001-99-4

Número da Comissão das Enzimas

EC 3.1.27.5 transferred to EC 4.6.1.18

Tipo de molécula

Enzyme

Product Line

Produtos biológicos

Armazenamento e envio

Concentração

from Bovine Pancreas; ≥80 Kunitz U/mg enzyme powder

Reconstituição

Reconstitute in 50 mM Tris-HCl (pH 7.4), 50% Glycerol.

Condições de armazenamento de armazenamento

Store at -20°C,Avoid repeated freezing and thawing

Enviado em

Ice chest + Ice pads

Estabilidade e armazenamento

Store at -20℃ long term (24 months). Upon reconstitution, it is recommended to aliquot. Avoid freeze/thaw cycle.

Application Protocols

The following protocols are provided as general, literature/practice guidance for RNase A use and are not item-specific performance claims. Optimize for your samples and consult the CoA/Spec Sheet for lot-specific details.

1. RNA removal during plasmid miniprep (general):

• Add RNase A to the resuspension buffer (e.g., 50–100 μg/mL) before cell lysis.
• Proceed with alkaline lysis and neutralization per kit/protocol. RNase A degrades RNA released during lysis, reducing viscosity and improving DNA purity.

2. Post-extraction DNA cleanup (general):

• To cleared DNA solution, add RNase A to a final concentration of ~10 μg/mL.
• Incubate 15–30 minutes at 37°C.
• Purify DNA by ethanol/isopropanol precipitation or spin column as appropriate.

3. RNA structure probing (general):

• Fold RNA in desired buffer (e.g., 10 mM HEPES pH 7.5, 50 mM KCl, MgCl2 as required).
• Add RNase A at limiting dilutions to achieve partial cleavage; incubate briefly at room temperature or 25–37°C.
• Quench with denaturant and analyze fragments by denaturing PAGE or capillary electrophoresis.

4. Removal of RNA from protein samples (general):

• Add a small amount of RNase A to viscous lysates to degrade RNA and aid filtration/chromatography.
• Incubate 15–30 minutes at room temperature or 37°C; remove RNase A by downstream purification if required.

Always maintain RNase-free technique except where RNase activity is intentionally introduced.

Biological Roles

RNase A is a secreted ribonuclease from bovine pancreas (RNase A superfamily) with a physiological role in RNA digestion. The following reflects literature knowledge, not item-specific claims.

• Function (literature): Catalyzes endonucleolytic cleavage of single-stranded RNA at pyrimidines, central to degradation of dietary RNA in the gastrointestinal tract. Operates via a two-step transphosphorylation/hydrolysis mechanism using His12/His119 as catalytic acid/base.
• Structure–function features (literature): Four disulfide bonds confer exceptional thermal stability and permit refolding after denaturation. Lys41 and active-site pocket architecture stabilize the transition state and promote specificity.
• Family and inhibition (literature): Part of the RNase A superfamily found in vertebrates; mammalian ribonuclease inhibitor (RI), a cytosolic protein with leucine-rich repeats, binds many RNase A family enzymes with femtomolar affinity, regulating extracellular/cytosolic RNase activity.
• Substrate preference (literature): Strong preference for single-stranded RNA; limited activity on RNA within stable double helices; can act on the RNA strand of RNA–DNA hybrids.
• Biotechnological context (literature): Benchmark model enzyme in protein folding, enzyme kinetics, and protein engineering studies; often used as a reference for disulfide bond formation/refolding research.

No biological therapeutic or clinical claims are made for this product. It is designated for research use only.

Buffer Applications

RNase A is typically used in aqueous buffers for RNA removal or structure probing. The following are literature/practice guidelines and not item-specific specifications.

• Stock solution preparation (general):
  • Dissolve in RNase-free water or 10 mM Tris-HCl, pH 7.5–8.0, with 15–100 mM NaCl.
  • Filter-sterilize (0.22 μm) if appropriate for long-term refrigerated storage; for −20°C storage, include 20–50% glycerol to prevent freezing and maintain activity.
• Heat treatment to minimize DNase contamination (general literature):
  • Incubate RNase A solution (e.g., 10 mM Tris-HCl, 15 mM NaCl, pH 7.5) at 95°C for 10–15 minutes; allow to cool slowly. RNase A retains activity while most DNases are inactivated. Validate for your application.
• Typical working uses (examples; literature/practice):
  • Plasmid miniprep: Include RNase A in resuspension buffer (e.g., 50–100 μg/mL) to degrade RNA during cell lysis, improving DNA purity and reducing viscosity.
  • DNA sample cleanup: Treat DNA solutions with a small amount of RNase A (e.g., 10 μg/mL) at 37°C for 15–30 minutes, then proceed with phenol-free cleanup (spin columns or precipitation) if needed.
  • RNA structure probing: Use defined RNase A dilutions in structure-mapping buffers (e.g., 10 mM HEPES, pH 7.5; 50 mM KCl; Mg2+ as required by the assay) and quench reactions with denaturants/chelators as per protocol.
• Compatibility notes (literature): EDTA does not inhibit RNase A (no metal dependence). Reducing agents that cleave disulfide bonds (e.g., high DTT at elevated temperature) can inactivate.

Always prepare and handle buffers in RNase-free conditions to avoid unintended contamination of RNA-sensitive workflows.

Green Alternatives

Enzymatic RNA removal using RNase A can serve as a greener alternative to solvent-intensive nucleic acid cleanup protocols.

• Greener practice vs. phenol/chloroform extraction (literature/practice):
  • Traditional RNA removal during DNA prep often uses acid phenol/chloroform, generating hazardous organic waste and posing operator exposure risks.
  • Incorporating RNase A into the buffer system reduces or eliminates organic extraction steps, lowering hazardous waste and improving lab safety.

Comparison (general):

• RNase A treatment
  • Pros: Aqueous, minimal hazardous waste, simple workflow integration, EDTA-compatible.
  • Cons: Must manage enzyme carryover; ensure DNase-free status for DNA applications; potential allergen/sensitizer handling.
• Phenol/chloroform extraction
  • Pros: Broadly effective for nucleic acid partitioning.
  • Cons: Toxic, corrosive, flammable waste; labor-intensive; disposal burden.

Additional green tips (literature/practice):

• Use concentrated RNase A stocks to minimize packaging and cold-chain volume; aliquot to reduce waste from degradation.
• Favor reusable, RNase-decontaminated glassware where appropriate; avoid single-use plastics when RNA integrity is not a concern.
• Substitute hazardous surface decontaminants with validated, lower-toxicity RNase deactivation solutions where effective.

Note: These are general sustainability considerations; validate any workflow changes for your specific samples and downstream assays.

Pharmaceutical Uses

This product is designated for research use only. The following points describe general, non-clinical roles of RNase A in bioprocessing contexts; they are not therapeutic claims and are not item-specific specifications.

• Process aid (literature/practice): RNases are sometimes used during manufacturing development or QC of biologics to reduce residual RNA in microbial lysates or viral vector preparations. Any such use requires appropriate process validation and regulatory-grade materials.
• Analytical workflows: Enzymatic removal of RNA can facilitate downstream analyses (e.g., DNA-based assays) by reducing interference from RNA.
• Excipient status: RNase A is not an excipient; its presence in final drug products is generally undesirable and subject to strict clearance requirements.

For any application in regulated manufacturing, a dedicated GMP-grade supply, full documentation, and validated clearance steps are necessary. This catalog item is intended strictly for research and laboratory use.

Physical Properties

Item-specific physicochemical specs (e.g., exact MW, extinction coefficient, pI) are not provided in the Product Data and may vary with lot and formulation.

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet. (Literature: RNase A monomer ~13.7 kDa)
• Solubility: High aqueous solubility (literature). Readily soluble in water and common biological buffers (Tris, phosphate); typically formulated in RNase-free water or buffer.
• Isoelectric point (pI): Literature ~9.3 (bovine RNase A).
• Extinction coefficient: Literature ε278 ~9,800 M−1·cm−1 (monomer) in water; sequence- and condition-dependent.
• Stability (literature): Thermally robust; retains/refolds activity after heating; stable over pH ~5–8. Strong denaturants (e.g., guanidinium chloride) and reducing agents that cleave disulfides can inactivate.
• Cofactors: None required (literature). Activity does not depend on metal ions; EDTA is compatible.
• Storage conditions (item-specific): Store at −20°C. Avoid repeated freezing and thawing (Product Data).

All numerical values above marked as literature are general references and not item specifications. For exact, lot-specific properties (concentration, formulation, activity), refer to the CoA/Spec Sheet.

Quality and Grades

This item is offered with multiple performance designations aimed at molecular biology workflows. The following are explanations of the grade terminology; they describe general expectations and are not additional specifications beyond the Product Data/CoA.

• DNase free (item-specific): Intended to be free of detectable DNase activity, enabling use in DNA purification where preservation of DNA integrity is essential. Practical implication: suitable for plasmid/miniprep buffers and genomic DNA workflows where RNA removal is needed.
• Native (item-specific): Protein is not recombinantly engineered or chemically modified; retains the native RNase A fold and catalytic properties typical of bovine pancreatic RNase A (literature description).
• Suitable for molecular biology (item-specific): Manufactured and handled to meet common molecular biology purity expectations (low contaminating nucleases/proteases, low bioburden) for research use.
• Bioactive, High Performance (item-specific): Indicates retention of enzymatic activity consistent with intended molecular biology applications. Exact unit activity and assay methods are not specified here; refer to CoA/Spec Sheet.
• ActiBioPure™ / EnzymoPure™ (item-specific brand designations): Internal quality branding denoting high purity and activity suitable for sensitive enzymatic workflows. Specific assay thresholds and impurity limits are not provided here; consult the CoA/Spec Sheet for lot-specific metrics.

Notes:

• UV cutoff, trace metals, stabilizers, or residual solvents: Not specified for this item; refer to CoA/Spec Sheet.
• For applications requiring regulated/GMP-grade materials, verify suitability; this listing is for research use only (Product Data).

Reaction and Applications

RNase A is a pyrimidine-specific endoribonuclease widely used across molecular biology workflows. The following uses and mechanistic notes are general (literature) and do not constitute item-specific performance specifications.

• Catalytic activity and specificity (literature):
  • Cleaves single-stranded RNA after cytidine and uridine residues via transphosphorylation to a 2′,3′-cyclic phosphate intermediate, followed by hydrolysis to 3′-phosphates.
  • Prefers single-stranded regions; activity on RNA–DNA hybrids is limited to the RNA strand.
  • No metal cofactors required; EDTA-compatible.
• Common applications (literature/practice):
  • Removal of RNA during plasmid and genomic DNA preparation (e.g., RNase A in resuspension buffers) to improve A260/280 ratios and downstream performance.
  • RNase protection assays and footprinting to probe RNA structure and RNA–protein interactions.
  • Cleanup of protein preparations contaminated with RNA to reduce viscosity and improve chromatographic behavior.
  • Post-reaction cleanup in reverse transcription or in vitro transcription workflows when selective RNA degradation is needed.
  • RNA mapping in combination with complementary nucleases (e.g., RNase T1, RNase V1) for structure probing.
• Practical tips (literature/practice):
  • Use RNase-free consumables and buffers; add RNase A last to minimize unintended contamination.
  • Typical working temperatures: ambient to 37°C. pH near neutral (e.g., 7.0–8.0) maximizes activity.
  • RNase inhibitors (e.g., mammalian ribonuclease inhibitor proteins) can inactivate RNase A family members; avoid introducing inhibitors when RNase activity is desired.
  • Heat treatment can inactivate potential DNase contaminants while preserving RNase A activity (see Buffer Applications for general conditions).

For unit definitions, specific activity, and recommended loadings for this item, refer to the CoA/Spec Sheet.

Reaction Conditions

General, literature-based conditions for RNase A-catalyzed RNA cleavage (not item-specific specifications):

• Temperature: Active from ambient to 37°C; many protocols run at 37°C for 15–60 minutes. Thermal stability is high; however, prolonged incubation at elevated temperatures with reducing agents can inactivate.
• pH: Optimal activity near neutral to slightly basic (approx. pH 6.5–8.0). Buffer choices include Tris-HCl, phosphate, or HEPES.
• Ionic strength: Moderate salt (e.g., 50–150 mM NaCl or KCl) often stabilizes the enzyme and reduces nonspecific interactions.
• Cofactors: None required. EDTA is compatible; divalent cations are not necessary for catalysis.
• Denaturants/detergents: High concentrations of chaotropes (guanidinium, urea) and ionic detergents (SDS) denature/inactivate. Nonionic detergents at trace levels are generally tolerated.
• Inhibitors: Mammalian ribonuclease inhibitor proteins potently inhibit RNase A family enzymes; sulfhydryl-reactive agents that stabilize RI can indirectly suppress RNase A activity in mixtures containing RI.
• Substrate scope: Single-stranded RNA, with preference for sites 3′ of cytidine/uridine. RNA–DNA hybrids are cleaved at the RNA strand.
• Typical workups: Following digestion, enzymatic reactions may be stopped by adding denaturants (e.g., SDS, formamide) and heat, or by using spin-column cleanup/precipitation.

For this product’s recommended working concentrations and activity units, consult the lot-specific CoA/Spec Sheet.

Safety and Handling

• GHS classification, signal word, pictograms, and H-statements: Not specified for this item; refer to SDS for authoritative information.
• General hazards (literature/best practice):
  • Enzyme proteins can be respiratory or skin sensitizers; avoid creating aerosols or dust. Handle in a fume hood or biosafety cabinet if procedures could aerosolize.
  • May cause irritation to eyes, skin, and respiratory tract upon contact or inhalation of powders/sprays.
• PPE recommendations: Lab coat, safety glasses, and appropriate disposable gloves. Use respiratory protection if aerosol generation is likely and engineering controls are insufficient.
• Safe handling:
  • Work on clean, RNase-free surfaces to avoid environmental RNase contamination of sensitive RNA workflows.
  • Avoid repeated freeze–thaw cycles (Product Data). Aliquot upon first thaw.
  • Prevent contact with strong oxidizers and reducing agents that may denature protein; maintain recommended storage temperature.
• First aid (summary; defer to SDS):
  • Inhalation: Move to fresh air; seek medical attention if symptoms persist.
  • Skin/eye contact: Rinse with water for several minutes; remove contaminated clothing; seek medical advice if irritation develops.
  • Ingestion: Rinse mouth; seek medical advice.
• Spills and disposal: Absorb small spills with inert material, clean with detergent, and dispose according to institutional and local regulations for proteinaceous lab waste.

Always consult the SDS for this specific product and lot for definitive hazard and response guidance.

Solvent Selection

RNase A is a water-soluble protein enzyme used in aqueous systems. Organic solvent selection is generally not applicable. Consider the following buffer-related guidance (literature/general):

• Preferred medium: RNase-free water or biological buffers (e.g., Tris-HCl, phosphate, HEPES) at near-neutral pH. Ionic strength (e.g., 50–150 mM NaCl) can enhance stability and reduce nonspecific binding.
• Additives for stability: 10–50% glycerol can help maintain activity during frozen storage and minimize freeze–thaw damage. Small amounts of nonionic detergents (e.g., 0.01% Tween-20) are sometimes used to reduce surface adsorption.
• Incompatible conditions: Strong reducing agents (that disrupt disulfides) and high concentrations of chaotropes can denature/inactivate RNase A. High concentrations of anionic detergents (e.g., SDS) will inactivate under denaturing conditions.
• Comparison with alternatives:
  • RNase A vs. RNase T1: RNase A cleaves at pyrimidines (C,U); RNase T1 cleaves after guanosine. Choice depends on mapping/processing needs.
  • Enzymatic RNA removal vs. solvent extraction: Using RNase A to remove RNA during DNA prep can reduce or eliminate hazardous phenol/chloroform steps.

For sample preparation and workflows, prioritize RNase-free aqueous buffers and avoid organic solvents that destabilize proteins.

Storage and Reconstitution

• Storage (item-specific): Store at −20°C. Avoid repeated freezing and thawing (Product Data). Shipped with cold packs.
• Aliquoting: Upon first thaw, divide into single-use aliquots to prevent activity loss from freeze–thaw cycles.
• Formulation: Not specified for this item; refer to CoA/Spec Sheet.
• Reconstitution (general literature guidance):
  • If supplied lyophilized: Reconstitute in RNase-free water or buffer (e.g., 10 mM Tris-HCl, pH 7.5–8.0; 50–100 mM NaCl) to the desired concentration. Mix gently; avoid foaming.
  • Optional stabilizers for frozen storage: Add sterile glycerol to 20–50% (v/v) to permit storage at −20°C without freezing; store in RNase-free microtubes.
• Handling: Keep on ice during use. Avoid prolonged exposure to room temperature. Do not vortex vigorously; invert or flick to mix.
• Shelf life: Not specified for this item; refer to CoA/Spec Sheet.

For any lot-specific concentration, unit activity, and buffer composition, consult the CoA/Spec Sheet and SDS.

Structure and Identity

RNase A is a well-characterized ribonuclease from bovine pancreas, belonging to the RNase A superfamily. It is a single-chain globular protein enzyme that catalyzes cleavage of RNA at pyrimidine residues.

• SKU: R1522663
• Product name: RNase A
• CAS: 9001-99-4
• PubChem CID: 9369
• InChIKey: Not applicable for proteins
• SMILES: Not applicable for proteins
• Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet. (Typical literature value for bovine RNase A monomer ~13.7 kDa)
• Source/type: Protein enzyme (pancreatic ribonuclease; RNase A family) — general description (literature)
• Structural features (literature):
  • Single polypeptide chain with four disulfide bonds forming a compact, highly stable fold.
  • Active-site residues include His12 and His119 (general acid–base catalysis) and Lys41 (stabilization of the transition state).
  • No prosthetic group or metal cofactor required; catalytic activity is intrinsic to the protein.
• 2D/primary structure description (literature): Linear amino acid sequence (~124 residues) folded into a compact globular architecture with α-helices and β-strands; disulfide bonds constrain the fold; the catalytic cleft binds single-stranded RNA.

Notes: Item-specific sequence, exact mass, or isoform details are not provided in the Product Data; consult CoA/Spec Sheet if needed.

Synthetic Utility

RNase A is a protein enzyme and is not used as a small-molecule building block or reagent for organic synthesis. Therefore, conventional synthetic utility (e.g., cross-coupling, protecting-group chemistry) does not apply.

Relevant laboratory utility (literature/practice):

• Biocatalysis in nucleic acid processing: Selective endonucleolytic cleavage of RNA at pyrimidines enables analytical mapping and cleanup steps in nucleic acid workflows.
• Protein engineering benchmark: RNase A is a classic model for protein folding/disulfide formation studies; used to test refolding strategies, oxidative folding buffers, and stabilization approaches — insights that may inform design of protein-based biocatalysts.

For chemical synthesis needs, refer to appropriate small-molecule reagents and catalysts; RNase A’s relevance is confined to enzymology and nucleic acid processing.

Target Specificity

Not applicable. This product is an enzyme (RNase A), not an antibody or affinity reagent. There are no antigen/epitope targets, clone designations, or species reactivity parameters associated with this item.