Ribonuclease A - Assay

Source:
Bovine Pancreas
CAS:
9001-99-4
EC:
3.1.27.5

Ribonuclease A Assay

Method
Standardization of ribonuclease activity has been difficult due to varying rates at which reactions occur as well as to the significant differences in nucleotide patterns in RNA isolated from biological sources. Crook et al. (1960) have published an assay using a synthetic substrate, cytidine 2', 3'-phosphate. Zimmerman and Sandeen (1965) described a sensitive assay using polycytidylic acid.

The method of Kalnitsky et al. (1959) is used at Worthington. The rate of hydrolysis of yeast RNA at pH 5.0 is determined by measuring the amount of acid soluble oligonucleotide released under defined conditions. One unit causes an increase in absorbance of 1.0 at A260 at 37°C and pH 5.0 under the specified conditions.

Reagents
  • 0.10 M Sodium acetate buffer, pH 5.0
  • 25% Perchloric acid containing 0.75% Uranyl acetate
  • 1% Worthington yeast RNA in 0.10 M sodium acetate, pH 5.0. Equilibrate to 37°C prior to assay.
Enzyme

Prepare stock solution at 1 mg/ml in reagent grade water. Immediately prior to assay, dilute further to 2, 4, and 6 micrograms per milliliter in 0.10 M sodium acetate, pH 5.0.

Procedure

Pipette one ml of respective enzyme dilution into centrifuge tubes. Include a blank containing one ml of 0.10 M sodium acetate buffer, pH 5.0. Incubate all tubes at 37°C for 5-8 minutes. At timed intervals, add one ml of 1% RNA. Incubate each tube exactly 4 minutes and stop reaction by the addition of one ml of uranyl acetate-perchloric acid solution. Transfer to an ice bath and cool for 5 minutes. Clarify by centrifugation and dilute 0.1 ml of clear supernatant to 3.0 ml with reagent grade water. Read A260 versus blank.

Calculation

Ribonuclease A Products

Description
Activity
Code
Cat. #
Size
Price
Description
Ribonuclease A, DNase & Protease Free
Source:
Bovine Pancreas
Molecular Biology Grade. A solution containing approximately 5 mg/ml in 50% glycerol. Prepared specifically for use in purifying DNA plasmids. Each lot is assayed for DNase and protease.
Store at 2-8°C or -20°C.
Activity
≥2,000 units per mg protein
Code
RPDF
LS002131
1 mg
$29.00
LS002132
5 mg
$95.00
LS002130
Bulk
---
Description
Ribonuclease A, Purified
Source:
Bovine Pancreas
A lyophilized preparation equivalent to Code: RASE. Contains some aggregates as a result of lyophilization but shows no difference in specific activity.
Store at 2-8°C. PROTECT FROM MOISTURE.
Activity
≥3,000 units per mg dry weight
Code
RAF
LS005649
25 mg
$76.00
LS005650
100 mg
$237.00
LS005655
Bulk
---
Description
Ribonuclease A, Purified Solution
Source:
Bovine Pancreas
Chromatographically purified. Aggregate-free. (Monophoretic on native gel electrophoresis.) A liquid in 100mM phosphate buffer containing 0.1% v/v phenol as a preservative. Note: The presence of 100mM phosphate makes this preparation unsuitable for heat treatment.
Store at -20°C.
Dry Ice required
Activity
≥3,000 units per mg protein
Code
RASE
LS005677
25 mg
$60.00
LS005679
100 mg
$152.00
LS005681
Bulk
---
Description
Ribonuclease A
Source:
Bovine Pancreas
A chromatographically purified, lyophilized powder.
Store at 2-8°C. PROTECT FROM MOISTURE.
Activity
≥2,500 units per mg dry weight
Code
R
LS003431
200 mg
$106.00
LS003433
1 gm
$440.00
LS003435
Bulk
---
Description
Ribonuclease B
Source:
Bovine Pancreas
A partially purified preparation containing a mixture of RNase A and RNase B. A soluble, dialyzed lyophilized powder.
Store at 2-8°C.
Activity
≥1,000 units per mg dry weight
Code
RB
LS005710
100 mg
$113.00
LS005715
Bulk
---
Description
Ribonuclease A, Recombinant, Protease & DNase Free, Animal Free/AF
Source:
Pichia pastoris
Recombinant bovine pancreatic Ribonuclease A produced in Pichia pastoris. Chromatographically purified, free of animal derived components, DNases and Proteases. Supplied as lyophilized powder. 
Store at 2-8°C. PROTECT FROM MOISTURE.
Code
RRA1
LS01506
10 ku
$175.00
LS01508
25 ku
$350.00
LS01510
Bulk
---
Description
Ribonuclease A, Recombinant, Animal Free/AF
Source:
Pichia pastoris
Recombinant Bovine pancreatic Ribonuclease A produced in Pichia pastoris, Animal Free/AF, Bioprocess grade. For the removal of RNA in bioprocessing applications. May contain DNases and Proteases. Supplied as a lyophilized powder.
Store at 2-8°C. PROTECT FROM MOISTURE.
Code
RRA2
LS01516
Bulk
---
LS01512
100 mg
$115.00
LS01514
1000 mg
$525.00