Navigation Links
Measuring RNase Activity A Real-time Kinetic Analysis


The RNaseAlert QC System, when used in conjunction with a fluorometer capable of kinetic analysis, provides a rapid and ultra-sensitive assay for determining RNase A specific activity. The RNaseAlert QC System also detects a variety of other nucleases, including RNase A, T1, RNase I, micrococcal nuclease, S1 nuclease, mung bean nuclease, and Benzonase. In addition to detecting nuclease activity, this kit is useful for standardizing activities of these enzymes.


Sources of RNase

Ribonucleases (RNases) are ubiquitous in the environment, and they are present in relatively high concentrations in many biological materials. For example, the pancreas is rich in RNase (>1 mg RNase/1 g tissue) and is the source for most commercially produced RNase A (and its glycosylated derivative, RNase B).

RNase A is an endonuclease that cleaves 3' of C and U residues. It is commonly used in molecular biology applications such as the removal of contaminating RNA from DNA preparations and ribonuclease protection assays (RPA). Ambion sells an RPA grade RNase A (Cat #2272) that is equivalent to molecular biology grade RNase A from other suppliers, as well as affinity-purified RNase A (Cat #2270 and #2271), which is recommended when the absence of DNase and other nonspecific nuclease activities is essential.


Measuring Specific Activity

Recently, Ambion introduced the RNaseAlert QC System, a quantitative fluorometric high-throughput assay for the detection of contaminating RNases. The assay consists of an oligonucleotide substrate that contains a fluorophore on one end and a quencher on the other. Once cleaved by RNase, the fluor and quencher are separated. When excited with 490 nm light, the fluor emits a bright green signal that can be quantitated with a fluorometer (Figure 1).

Figure 1. Schematic of the RNaseAlert QC System.

Are Your Enzyme Units Uniform?

During routine protein purification some proportion of the enzyme population is inevitably inactivated. Therefore, the proportion of active enzyme is routinely quantitated by measuring the specific activity, the amount of active enzyme per mg of the total protein. In 1950, M. Kunitz developed the first spectrophotometric assay for measuring DNase I activity. The assay was based on the increase in A260 at pH 5.0 and 25C caused by the degradation of a DNA substrate. Kunitz also recognized that the rate of increase of A260 under the proper conditions, is both linear and constant and that the linear rates were proportional to the concentration of the active enzyme.

The Kunitz unit has since become the standard unit of measurement for enzymatic activity. However, the original Kunitz assay was performed under conditions that are not optimal for many enzymes, so most manufacturers have developed their own unit assays. Therefore, it is important to look closely at both the manufacturer's unit definition as well as the assay conditions to determine how an enzyme will function in your specific application.

One Kunitz unit (K) produces a change in A260 of 0.001/min/ml at pH 5.0, 25C or 1 unit hydrolyzes a substrate at a rate constant of K = 1 at pH 5.0, 25C.

Here we report the steady-state kinetic analysis of RNase A activity using the RNaseAlert QC System. RNase A (700 U/mg, 10 ng/ml) was serially diluted and incubated with a large excess of substrate such that the amount of input enzyme was proportional to the rate of the reaction. A baseline was established by monitoring substrate alone in RNaseAlert Buffer. After several minutes, experimental samples containing 0.5 pg, 5.0 pg and 50 pg of RNase A were added. The rate of increase in fluorescence was measured in real-time at 37C in 2 minute increments for 1 hour. Figure 2 shows reaction curves plotted for 3 concentrations of enzyme: 50 pg, 5.0 pg, and 0.5 pg. After enzyme addition, the rate of increase in fluorescence was constant for several minutes. Under these conditions, the reaction is at equilibrium and the rate of increase in fluorescence (the slope of the line) can be used to calculate the initial rate (or velocity) of the reaction. As expected, when initial velocities were compared (see legend, Figure 2), there was a 10-fold difference between each 10-fold serial dilution. These data demonstrate that the dilutions maintain the mathematical proportionality predicted for steady-state kinetic analysis.

Figure 2. Kinetics of RNaseAlert Cleavage by RNase A. Using conditions of substracte excess, the rate of increase in fluorescence (the slope of the line) was used to calculate the initial reaction rates for three concentrations of RNase A. The initial velocities were 0.005 RFU/min, 0.054 RFU/min, and 0.5 RFU/min for 0.5 pg, 5.0 pg and 50 pg Rnase A, respectively.


In the Kunitz assay, the linear rate of increase of A260 is proportional to the concentration of the enzyme in solution. The activity of the enzyme is expressed in terms of the slope of the plotted curve of absorbance versus time. Likewise, when tested with the RNaseAlert QC System, the early, linear phase of the reaction yields an increase in relative fluorescence units (RFU) that is proportional to the concentration of enzyme in solution. Therefore, the RNaseAlert QC System, when used in conjunction with a fluorometer capable of kinetic analysis, provides a rapid and ultra-sensitive assay for quantitative measurements of RNase A activity. Since this assay also detects a variety of other nucleuses (RNase T1, RNase I, micrococcal nuclease, S1 nuclease, mung bean nuclease, and Benzonase), it should also be useful for standardizing their specific activity.


References

  1. Kunitz, M (1950) Crystalline Deoxyribonuclease I. Isolation and general properties, spectrophotometric method for the measurement of desoxyribonuclease activity. J. Gen. Physiol. 33: 349-362.
  2. 2. Crook, E, Mathias, A Rabin, B (1960) Spectrophotometric assay of bovine pancreatic ribonuclease by the use of cytidine 2',3'-phosphate. Biochem. J. 74: 234.


back to top


Ordering Information
Cat# Product Name Size 1966 RNaseAlert QC System 5 x 96 assays 2270 RNase A (Affinity purified) 1 mg/ml 200 g 2271 RNase A (Affinity purified) 1 mg/ml 1 mg 2272 RNase A (RPA grade) 1 mg/ml 1 mg
'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Measuring multiple apoptosis parameters with the Agilent 2100 bioanalyzer
2. Measuring Gene Silencing Effects by RT-PCR Without RNA Isolation
3. Measuring Intracellular Enhanced Green Fluorescent Protein With the VersaFluor Fluorometer
4. Measuring siRNA-mediated knockdown of IL-8 mRNA
5. Protector RNase Inhibitor
6. Protector RNase Inhibitor Enhance the Protection of RNA against Degradation
7. RNase Protection Assay (RPA) Using DIG-Labeled RNA Probes
8. Inducing RNAi with siRNA Cocktails Generated by RNase III
9. Dicer vs. RNase III for Preparation of siRNA Cocktails
10. Combat RNase Contamination in the Lab
11. RNase Activity in Mouse Tissue: Classification, Hierarchy, and Methods for Control
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Measuring RNase Activity Real time Kinetic Analysis

(Date:7/30/2014)... July 30, 2014. Applied DNA ... that provides DNA-based authentication and security solutions and services, ... of Life Sciences, a newly created position, with a ... will lead APDN’s drive to build its business in ... healthcare customers, provide guidance to APDN’s R&D efforts, and ...
(Date:7/30/2014)... Fla. , July 30, 2014  Dyadic ... biotechnology company whose patented and proprietary technologies are ... and other proteins for the bioenergy, bio-based chemical, ... appointment of Thomas "Tom" Dubinski as Vice President ... for leading the financial and information technology operations ...
(Date:7/30/2014)... Edwardsville, IL (PRWEB) July 30, 2014 ... Dr. Arun Athmanathan will present his findings on ... of Energy conference this week in Washington, D.C. ... Department of Energy conference is a testament to ... Director John Caupert said. “Thanks to the foresight ...
(Date:7/30/2014)... LayerBio, Inc., an MIT spinoff developing novel drug ... seed financing from friends and family investors. Proceeds from ... novel sustained-release product for ophthalmology. , "We are grateful ... to this important work," said Dr. Ken Mandell, Founder ... areas in which innovations in drug delivery have already ...
Breaking Biology Technology:Dr. Michael E. Hogan, Ph.D., Joins Applied DNA Sciences as Vice President, Life Sciences 2Dr. Michael E. Hogan, Ph.D., Joins Applied DNA Sciences as Vice President, Life Sciences 3Thomas Dubinski joins Dyadic as Chief Financial Officer 2Thomas Dubinski joins Dyadic as Chief Financial Officer 3Thomas Dubinski joins Dyadic as Chief Financial Officer 4NCERC at SIUE Researcher Takes Stage at Department of Energy Conference 2NCERC at SIUE Researcher Takes Stage at Department of Energy Conference 3
... on Thursday, October 25, 2007 at ... ... Keryx Biopharmaceuticals,Inc. (Nasdaq: KERX ), a biopharmaceutical company focused on ... treatment of life-threatening,diseases, including diabetes and cancer, today announced its results ...
... Biosciences, Inc. Presents Initial Data on RDEA119 at ... and Cancer Therapeutics -, SAN FRANCISCO, Oct. ... a company focused on the discovery and development ... cancer and,inflammatory diseases, today presented preclinical data on ...
... Md., Oct. 24 MedImmune, Inc. announced,today that ... recognized the company for its employees, exceptional commitment ... Volunteer Award,for a large business. "As the ... as an outstanding corporate citizen whose employees strive ...
Cached Biology Technology:Keryx Biopharmaceuticals, Inc. Announces Third Quarter 2007 Financial Results 2Keryx Biopharmaceuticals, Inc. Announces Third Quarter 2007 Financial Results 3Keryx Biopharmaceuticals, Inc. Announces Third Quarter 2007 Financial Results 4Keryx Biopharmaceuticals, Inc. Announces Third Quarter 2007 Financial Results 5Keryx Biopharmaceuticals, Inc. Announces Third Quarter 2007 Financial Results 6New Oral MEK Inhibitor, RDEA119, Shows Favorable Anti-Tumor Properties 2New Oral MEK Inhibitor, RDEA119, Shows Favorable Anti-Tumor Properties 3New Oral MEK Inhibitor, RDEA119, Shows Favorable Anti-Tumor Properties 4
(Date:7/30/2014)... climate change on the world,s animal and plant ... to a new paper by a research team ... and other organizations. The authors claim that most ... when it comes to climate change. , ... on the "direct" threats of changing temperatures and ...
(Date:7/30/2014)... San Antonio Life Sciences Institute (SALSI), a joint venture ... and The University of Texas Health Science Center at ... Initiative Pilot Program. The winners, Emily Boice from UTSA ... receive $25,000 for their project titled, "Novel engineered ferritins ... environment." , Boice and Huang,s research is focused on ...
(Date:7/30/2014)... University associate professor Andrew S. Mount, performed cutting-edge research ... for novel anti-fouling paint for ships and boats and ... The team,s findings, published in Nature Communications ... that attaches to a wide variety of surfaces using ... an underwater heavy-duty adhesive. , "In previous research, we ...
Breaking Biology News(10 mins):Conservation scientists asking wrong questions on climate change impacts on wildlife 2Study: Marine pest provides advances in maritime anti-fouling and biomedicine 2
... hockey goalie, and two opposing players are breaking in alone ... of the linesman skating in on your left, but pay ... the two approaching players. As the action unfolds, how is ... splitting your focus of attention into multiple "spotlights?" Are you ...
... by researchers at Columbia University,s Mailman School of Public ... quality regulations proposed or recently adopted by the U.S. ... standards for reducing dangerous emissions of mercury and other ... implementing the new regulations is estimated to be about ...
... , The National Institute of Standards and Technology (NIST) ... carbon nanotube soot. Distantly related to the soot in ... is the primary industrial source of single-wall carbon nanotubes, ... NIST material offers companies and researchers a badly needed ...
Cached Biology News:How do we split our attention? 2New EPA air quality rules outweigh costs and provide major health and environmental benefits 2NIST releases first certified reference material for single-wall carbon nanotubes 2NIST releases first certified reference material for single-wall carbon nanotubes 3
...
...
with GlutaMAXIL-glutamine...
... PURIFIED GOAT ANTI-MOUSE IgG+IgA+IgM ... conjugated with highly purified ... The antibody has been ... rat, fetal bovine, horse, ...
Biology Products: