商品编号


Y9370L



品牌


Enzymatics



公司


Enzymatics,inc



公司分类


New Products



Size

1,250 U

商品信息

Thermo
stable Pyrophosphatase


Product Description

Thermo
stable pyrophosphatase is a recombinant enzyme from Sulfolobus acidocaldarius which catalyzes the Mg-dependent reaction of P
2
O
7
-4
+ H
2
O → 2HPO
4
-2
. It is has a low Km (5.4 uM) for pyrophosphate, is active between pH 7 and 9, has an optimal temperature for activity at 75°C and is functional under PCR conditions.

Source of Protein

A recombinant
E. coli
strain carrying the
Thermo
stable Pyrophosphatase gene from S.
acidocaldarius
.

Unit Definition
One unit is the amount of enzyme that will liberate 1 ?mol of phosphate per minute from inorganic pyrophosphate at 75°C and pH 8.5

Molecular Weight
19,381 Daltons

?



Quality Control Analysis

Unit

Activity
The assay is based on that described by Taussky and Shorr (4). Briefly, enzyme dilutions are added to 30mM Tris HCl pH 8.5, 1.5 mM MgCl2 and 1.5mM sodium pyrophosphate. After a 10 min incubation at 75&def;C, the product formed, 2- orthophosphate, is reacted with ammonium moly
BD
ate to form phosphomoly
BD
ic acid. The phosphomoly
BD
ic acid is then reduced by ferrous sulfate under weak acidic conditions to form a blue color, the absorbance of which is measured at 660nm. The amount of product formed is extrapolated from a phosphate standard curve generated from the ammonium molydate/ferrous sulfate reaction.

Protein

Concentration (OD
280


)
is determined by OD
280
absorbance.

Physical Purity
is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the protein of interest band in the diluted sample.

Single-Stranded

Exonuclease
is determined in a 50 ?L reaction containing a r
ADI
olabeled single-stranded DNA substrate and 10 ?L of enzyme solution incubated for 4 hours at 37°C.

Double-Stranded Exonuclease
is determined in a 50 ?l reaction containing a r
ADI
olabeled double-stranded DNA substrate and 10 ?L of enzyme solution incubated for 4 hours at 37°C.

Double-Stranded Endonuclease
is determined in a 50 ?L reaction containing 0.5 ?g of plasmid DNA and 10 ?L of enzyme solution incubated for 4 hours at 37°C.

E.coli
16S rDNA Contamination
is evaluated using 5 ?L
r
eplicate samples of enzyme solution 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.

Supplied in

10 mM Tris-HCl pH 7.5
50 mM NaCl
50% Glycerol
1 mM DTT
0.1 mM EDTA


View PDF Poster Instructions FAQ


Product Information



Thermo
stable Pyrophosphatase


Part Number
Y9370L
Price
$230
Concentration
2,000 U/ml
Unit Size
1,250 U


SDS

Available on request








Product Specification*


Storage Temperature
-25 to -15°C


Test

Units Tested

Specification


SDS Purity
n/a
>95%
Specific Activity
n/a
3,500 U/mg
SS Exonuclease
50
<1.0% Released
DS Exonuclease
50
<1.0% Released
DS Endonuclease
50
No Conversion
E.coli
DNA Contamination
50
<10 copies





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

References


Sulfolobus acidocaldarius inorganic pyrophosphatase: structure,
Thermo
st
ABI
lity, and effect of metal ion in an archael pyrophosphatase. Lepp?nen VM, Nummelin H, Hansen T, Lahti R, Sch?fer G, Goldman A. Protein Sci. 1999. 8(6):1218- 31.

The extreme
Thermo
stable pyrophosphatase from Sulfolobus acidocaldarius: enzymatic and comparative biophysical characterization. Hansen T, Urbanke C, Lepp?nen VM, Goldman A, Brandenburg K, Sch?fer G. Arch Biochem Biophys. 1999. 363(1):135-47.

Purification, cloning, and sequencing of archaebacterial pyrophosphatase from the extreme
Thermo
acidophile Sulfolobus acidocaldarius. Meyer W, Moll R, Kath T, Sch?fer G. Arch Biochem Biophys. 1995. 319(1):149-56.

A microcolorimetric method for the determination of inorganic phosphorus. Taussky HH, Shorr E. J Biol Chem. 1953 Jun;202(2):675-85.




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.