Download rAPid Alkaline Phosphatase - Sigma

For life science research only. Not for use in
diagnostic procedures. FOR IN VITRO USE ONLY.
rAPid Alkaline Phosphatase
Orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1
Cat. No. 04 898 133 001
Cat. No. 04 898 141 001
1000 units
5000 units
y Version 2.0
Nov. 2006
Store at ⫺15 to ⫺25°C
1.
What this Product Does
Enzyme Characteristics
Number of Tests
1 kit is designed for
• 1000 dephosphorylation reactions (Cat. No. 04 898 133 001)
• 5000 dephosphorylation reactions (Cat. No. 04 898 141 001)
with a final reaction volume of 20 ␮l each.
Parameter
Description
Source
Recombinant alkaline phosphatase from bovine intestine (1) expressed in Pichia pastoris
Molecular
weight
56 kD (by SDS-PAGE, monomer)
Pack Contents
Subunits
homodimer (Zn2+ is essential for activity)
Label
Contents / Function
Unit Definition One unit of rAPid Alkaline Phosphatase is the enzyme
activity which hydrolyzes 1 ␮mol of 4-nitrophenyl
phosphatase in 1 min at 37°C under assay conditions.
rAPid Alkaline Phos- 0.5 M Tris/HCl, 1mM EDTA, pH 8.5 (20°C)
phatase Buffer, 10⫻
conc.
Volume
Activity
1 U/␮l
rAPid Alkaline Phos- • 1000 U (Cat. No. 04 898 133 001)
phatase
• 5000 U (Cat. No. 04 898 141 001)
Specific
Activity
Approx. 1U/␮g according to (2) and (3).
L See data label for lot-specific values.
Storage and Stability
If stored at ⫺15 to ⫺25°C the product is stable through the expiration
date printed on the label.
L The product is shipped on dry ice.
L Storage buffer: 25 mM Tris/HCl; 1mM MgCl2; 1mM ZnCl2; 50 %
Glycerol (v/v); pH approx. 7.6 (4°C)
Specificity
Alkaline Phosphatase catalyzes the hydrolysis of
numerous phosphate esters, such as esters of primary
and secondary alcohols, saccharides, cyclic alcohols,
phenols and amines. Phosphodiesters do not react.
The enzyme hydrolyzes inorganic pyrophosphate. The
kinetic properties of the enzyme depend on many factors, such as purity of enzyme, concentration of
enzyme in the assay, buffer, pH etc.
Application
rAPid Alkaline Phosphatase is used to dephosphorylate 5' ends of
DNA and RNA. Alkaline phosphatase treatment prevents self-ligation
of fragments by removing the 5'-phosphoryl termini required by
ligases; this feature is of major importance in cloning strategies to
decrease vector background. rAPid Alkaline Phosphatase can also be
employed to dephosphorylate proteins.
Use rAPid Alkaline Phosphatase in numerous applications, including:
• Removal of 5'-phosphoryl groups from nucleic acids
• Preparation of templates for 5'-end labeling
• Clean-up of PCR products by removal of dNTPs
• Dephosphorylation of proteins
rAPid Alkaline Phosphatase is very rapidly inactivated by heating at
75°C for 2 min. No additional purification steps are required after
restriction and dephosphorylation. The dephosphorylated DNA can
directly be used in a ligation reaction.
Reaction
Temperature
37°C
Inactivation
rAPid Alkaline Phosphatase is inactivated by incubation at 75°C for 2 min.
Animalderived
additives
none
1106.04951514001
Assay Time
The assay time depends on the nature of the DNA ends to be dephosphorylated:
• Up to 1 ␮g DNA with blunt or sticky 5' overhang ends: 10 min
dephosphorylation at 37°C
• Up to 1 ␮g DNA with sticky 5' recessive ends: 30 min dephosphorylation at 37°C
Dephosphorylation is followed by 2 min inactivation at 75°C.
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2.
How to Use this Product
Standard Dephosphorylation Procedure
For standard dephosphorylation reactions, follow the procedure below.
L After restriction digestion with a Roche Applied Science restriction
enzyme you can use the DNA directly in the dephosphorylation
step. No additional purification steps are required.
Step
Action
쐃
• Thaw all necessary components.
• Briefly centrifuge all reagents before starting.
�
• Add the following reagents to a reaction vial:
Reagent
Volume
Final Concentration
vector DNA
x ␮l
up to 1 ␮g
rAPid Alkaline Phosphatase
Buffer, 10⫻ conc.
2 ␮l
1⫻
rAPid Alkaline Phosphatase
1 ␮l
1U
water, sterile deionized
add to
20 ␮l
Total Volume
20 ␮l
References
1 Manes, T. et al. (1998). Genetic Complexity, Structure and Characterisation of Highly Active Bovine Intestinal Alkaline Phosphatases. J. Biol. Chem. 273, 23353 – 23360.
2 Moessner, E. et al. (1980). Purification of human and bovine
alkaline phosphatase by affinity chromatography. Z. Physiol.
Chem. 361, 543.
3 Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal. Biochem. 72, 248-254.
4.
• Mix thoroughly and centrifuge briefly.
�
• Incubate DNA with blunt or sticky 5' overhang ends for
10 min at 37°C.
• Incubate DNA with sticky 5' recessive ends for 30 min at
37°C.
�
Inactivate the rAPid Alkaline Phosphatase for 2 min at
75°C.
쐄
Either use the dephosphorylation reaction mixture
directly in the following ligation reaction or store it at
⫺15 to ⫺25°C until further use.
3.
Absence of nick- 1 ␮g"supercoiled" pBR322 DNA is incubated with
ing activities
various amounts of rAPid Alkaline Phosphatase
in 50 ␮l of all different SuRE/Cut Buffers available
from Roche Applied Science. This shows no
decrease or degradation of the supercoiled
pBR322 DNA after agarose gel electrophoresis.
Supplementary Information
Ordering Information
Roche Applied Science offers a large selection of reagents and systems for life science research. For a complete overview of related
products, please visit and bookmark our home page, www.rocheapplied-science.com, and our Special Interest Sites including:
• PCR - Innovative Tools for Amplification: http://www.roche-appliedscience.com/pcr
• Mapping & Cloning: http://www.restriction-enzymes.com
Product
Pack Size
Restriction Enzymes
refer to Roche Applied Science home
page and catalogue
Agarose MP )
100 g
500 g
11 363 514 910
T4 DNA Ligase
100 U [1 U/␮l]
500 U [1 U/␮l
500 U [5 U/␮l]
10 481 220 001
10 716 359 001
10 799 009 001
T4 RNA Ligase
500 U
11 449 478 001
Agarose Gel DNA Extraction Kit
1 kit (max. 100 reactions)
11 696 505 001
High Pure PCR Product Purification Kit
Kit for 50 purifications 11 732 668 001
Kit for 250 purifications 11 732 676 001
Water, PCR-grade
25 ml (25 vials of 1 ml) 03 315 932 001
25 ml (1 vials of 25 ml) 03 315 959 001
100 ml (4 vials of 25
03 315 843 001
ml)
Additional Information on this Product
Product Description
rAPid Alkaline Phosphatase is isolated from bovine intestine and supplied as a recombinant enzyme expressed in the yeast, Pichia Pastoris.
The recombinant form ensures consistency and safety. rAPid Alkaline
Phosphatase catalyzes the dephosphorylation of 5' phosphates from
DNA and RNA, nucleotides, and proteins. Unlike calf intestinal phosphatase, rAPid Alkaline Phosphatase is rapidly, completely, and irreversibly inactivated by heat treatment for two minutes at +75°C. It is
therefore an excellent alternative to Shrimp Alkaline Phosphatase. In
addition, the enzyme is active in restriction enzyme buffers; therefore,
restriction enzyme digestion, dephosphorylation, enzyme inactivation,
ligation, or 5'-end labeling can be performed without purification
steps.
Background Information
DNA ligases join linear DNA fragments together via covalent bonds.
DNA ligation involves creating a phosphodiester bond between the 3'
hydroxyl group of one nucleotide and the 5' phosphate of another.
Ligation of DNA fragments is an essential step in many molecular biology techniques, including gene cloning and messenger RNA (mRNA)
fingerprinting. For efficient ligation, DNA strands must be prevented
from self-ligating (self-circularization and concatenation) by dephosphorylation of DNA ends. Alkaline phosphatase removes the 5'-phosphoryl termini required by ligases, preventing self-ligation of DNA and
decreasing background.
Quality Control
L See data label for lot-specific values.
1 ␮g ␭ DNA is incubated with rAPid Alkaline
Absence of
deoxyribonucle- Phosphatase for 16 hrs in 50 ␮l of all different
SuRE/Cut Buffers available from Roche Applied
ases
Science. This shows no degradation of ␭ DNA
after agarose gel electrophoresis.
Cat No.
Trademarks
HIGH PURE and SURE/CUT are trademarks of Roche.
Regulatory Disclaimer
For life science research only. Not for use in diagnostic procedures.
Contact and Support
To ask questions, solve problems, suggest enhancements or report new
applications, please visit our Online Technical Support Site at:
www.roche-applied-science.com/support
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Roche Applied Science
68298 Mannheim
Germany