商品编号


Y9240L



品牌


Enzymatics



公司


Enzymatics,inc



公司分类


RNA Enzymes



Size

20,000 U

商品信息

RNAse Inhibitor


Product Description

RNAse Inhibitor is an acidic, 52 kDa protein that is a potent non-competitive inhibitor of pancreatic-type ribonucleases such as RNase A, RNase B, and RNase C. The enzyme is provided as a fusion of the porcine RNAse Inhibitor gene with a proprietary, 22.5 kDa protein tag.

Source of Protein
A recombinant
E. coli
strain carrying the porcine RNAse Inhibitor gene.

Supplied in
20 mM Hepes-KOH
50 mM KCl
8 mM DTT
50% glycerol
pH 7.5 @ 25°C

Unit Definition
One unit is defined as the amount of enzyme required to inhibit by 50% the hydrolysis of cytidine 2′,3′-cyclic monophosphate by 5 ng of RNAse A. (1)



Quality Control Analysis

Unit Characterization Assay
Specific activity was determined using 1.1-fold serial dilution method. Dilutions of enzyme were made in 1X RNAse Inhibitor Reaction Buffer and added to 1000 ?L reactions containing 1mM cytidine 2′,3′-cyclic monophosphate, 1?g RNase A in a 1X reaction buffer containing 100mM Tris-Acetate, 1mM EDTA, pH 6.5. Absorbance at 286nm was observed at 20 second intervals during a 5minute reaction.

Protein Concentration (OD
280
) Measurement
A 2.0 ?L sample of enzyme was analyzed at OD
280
using a
Nanodrop
ND-1000 spectrophotometer standardized using a 2.0 mg/ml BSA sample (
Pierce
Cat #23209) and blanked with product storage solution. The observed average measurement of 3 replicate samples was converted to mg/mL using an extinction coefficient of 59,600 and molecular weight of 74,828 Daltons.

SDS-Page (Physical Purity Assessment)
2.0 ?L of concentrated enzyme solution was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW
Marker
and 2.0 ?L of a 1:100 dilution of the sample. Following electrophoresis, the gel was stained and the samples compared to determine physical purity. The acceptance criteria for this test requires that the aggregate mass of contaminant bands in the concentrated sample do not exceed the mass of the protein of interest band in the dilute sample, confirming greater than 99% purity of the concentrated sample.



Contamination Tests

Single-Stranded Exonuclease Activity
A 50 ?l reaction containing 10,000 cpm of a r
ADI
olabeled single-stranded DNA substrate and 10 ?L of enzyme solution incubated for 4 hours at 37°C resulted in less than 5.0% release of TCA-soluble counts.

Double-Stranded Exonuclease Activity
A 50 ?l reaction containing 5,000 cpm of a r
ADI
olabeled double-stranded DNA substrate and 10 ?L of enzyme solution incubated for 4 hours at 37°C resulted in less than 1.0% release of TCA-soluble counts.

Double-Stranded Endonuclease Activity
A 50 ?L reaction containing 0.5 ?g of pBR322 DNA and 10 ?L of enzyme solution incubated for 4 hours at 37°C resulted in no visually discern
IBL
e conversion to nicked circular DNA as determined by agarose gel electrophoresis.

Non-Specific RNAse Assay
Product was screened for non-specific RNAse contamination using the RNAse Alert kit, (Integrated DNA Technologies), following the manufacturer’s guidelines.

E.coli
16S rDNA Contamination Test

Replicate 5 ?L samples of enzyme solution were denatured and screened in a TaqMan qPCR assay for the presence of contaminating
E.coli
genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus. The acceptance criterion for the test is the threshold cycle count (C
t
) produced by the average of 3 replicate no template control samples. Based on the correlation between the no template control C
t
values, and standard curve data, the detection limit of this assay is <10 copies genome/sample.


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Product Information



RNAse Inhibitor


Part Number
Y9240L
Price
$363
Concentration
40,000 U/ml
Unit Size
20,000 U


SDS

Available on request








Product Specification*


Storage Temperature
-25 to -15°C


Test

Specification


Purity (SDS-PAGE)
>99%
SS Exonuclease
2000 U <5.0% released
DS Exonuclease
2000 U <1.0% released
DS Endonuclease
2000 U = No conversion
E.coli
DNA Contamination
2000 U <10 copies
RNAse Contamination
2,000 U = No Detectable non-specific RNase





* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.

References


Blackburn, P., 1979. Ribonulcease Inhibitor from Human Placenta: Rapid Purification and Assay. The Journal of
BIOLOG
ical Chemistry, Vol. 254, No. 24 pp 12484-12487.




Limitations of Use
This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for admi
NIST
ration to humans or animals. SDS sheets relevant to this product are available upon request.