Ribonuclease A (RNase A)
Order number: S5231.0100Shipping: shipped at RT, stored at -20°C
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Ribonuclease A is an endo-ribonuclease specifically cleaving single stranded RNA at the 3' side of pyrimidine bases (cytosine and uracil). RNAse A ist used to prepare RNA-free DNA, to digest non-hybridised regions of RNA-DNA hybrids and as a molecular weight marker. The pH optimum for RNAse A is between 7.0-7.5. RNAse A is inhibited by Diethylpyrocarbonate (DEPC) >, guanidinium salt (4M GuaSCN), beta-Mercaptoethanol, heavy metals, vanadyl-ribonucleoside complexes, RNAse-inhibitor from human placenta and by competitive DNA. RNAse A cleaves single and douple stranded RNA and RNA in RNA:DNA hybrids at low salt conditions (100 mM NaCl). At high salt conditions (>300 mM NaCl), RNAse A cleaves single-stranded RNA only. The enzyme can only be removed by Proteinase K digest and subsequent phenol extraction.
Elimination of DNAse activity:
Product S5231 (RNAse A (not certified DNAse free)) may contain DNAse activity. As RNAse A is heat stable it is recommended to heat inactivate DNAse activity before use.
10mg/mL RNAse A in 0.01 sodium acetate (pH5.2) have to be heated to 100°C for 15 minutes in a water bath. After 15 minutes, the RNAse shall remain in the water bath while the water cools down to room temperature. pH can be adjusted by addition of 0.1 time the volume of 1M Tris-HCl (pH7.4). After aliquotation of the now DNAse free RNAse A solution, each aliquote should be stored a -20°C.
NOTE: RNAse A precipitates if concentrated solutions are heated to 100°C at neutral pH!
Stock solution: Stock solutions are prepared at concentrations from 1 - 10mg/mL in 10mM Tris/HCl, pH7.5; 15mM NaCl or in 10mM Tris/HCl, pH7.5; 1mM EDTA, pH8.0 (TE buffer).
The recommended working concentration is 10µg/mL (removal of RNA from plasmid preparations; 1hr, RT) or 100ng/mL (preparation of 'blunt ends' of double-stranded cDNA).
Stability:RNase A aggregates during lyophilizing and storage. It has a high affinity to glas surfaces, which has to be taken into consideration. At neutral pH (e. g. in PBS pH 7.4) and high concentrations (>10mg/mL) the enzyme will precipitate. At +2°C to +8°C (lyophilized) it is stable for several years (dry storage), in solution (-20°C) several years or (+2°C to +8°C) several weeks.
• RNase protection assays • Remove unspecifically bound RNA • Analysis of RNA sequences • Hydrolyze RNA contained in protein samples • Purification of DNA
Activity: min. 70 kunits/mg
MW = approx. 13,700 Da
Product is isolated from BSE free source.
Optimal temperature: 60°C (activity range of 15–70°C); Optimal pH: 7.6 (activity range of 6–10); Inhibitors: ribonuclease inhibitor. Activators of RNase A: potassium and sodium salts.
application:RNase protection assays; Remove unspecifically bound RNA; Analysis of RNA sequences; Hydrolyze RNA contained in protein samples; Purification of DNA; Pancreatic RNase A specifically cleaves at the 3'-side of pyrimidine (uracil or cytosine) phosphate bonds
Unit Definition:1 unit corresponds to the amount of enzyme which hydrolyzes RNA at a rate constant k = 1 at 25°C and pH 5.0 (Kunitz-units), M. Kunitz, J. Biol. Chem. 164, 563 (1946).
Sicherheits Hinweise / Safety
Klassifizierungen / ClassificationEC-Nr: 232-646-6
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Cytotechnology. 2017 Feb; 69(1): 1–17. Published online 2016 Dec 1. doi: 10.1007/s10616-016-0021-z