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Calf Intestine Alkaline Phosphatase (CIAP)

#EF0341 Supplied with: 10X Reaction Buffer	200 u (1 u/µl) 1 ml
#EF0342 #EF0343 Both supplied with: 10X Reaction Buffer	5x200 u (1 u/µl) HC, 1000 u (10 u/µl) 5x1 ml
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EF0341, #EF0342, #EF0343 MSDS (English) MSDS (English-USA) MSDS (German)

 

Feature
Active in Fermentas buffers for restriction enzymes, PCR and RT.

Description
The Calf Intestine Alkaline Phosphatase (CIAP) catalyzes the release of 5'- and 3'-phosphate groups from DNA, RNA and nucleotides. Also this enzyme can remove phosphate groups from proteins.

Source
Calf intestine.

Molecular Weight
This enzyme is homodimer. It consists of two identical subunits of 65 kDa.

Applications

• Dephosphorylation of DNA and RNA (1), see Protocol for Dephosphorylation of DNA 5'-termini.
• Degradation of dNTPs in PCR mixture prior to sequencing of PCR products, see Protocol for PCR Product Clean-up (procedure according (2)).
• Dephosphorylation of proteins (3), see recommendations below.

Quality Control
The absence of endodeoxyribonucleases, exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests. Functionally tested for dephosphorylation of DNA 5'-termini.

Concentration
1 u/µl ; 10 u/µl, HC

Definition of Activity Unit
One unit of the enzyme hydrolyzes 1 µmol of 4-nitrophenylphosphate in 1 min at 37°C.
Enzyme activity is assayed in the following mixture: 1 M diethanolamine-HCl (pH 9.8), 0.5 mM MgCl₂, 10 mM 4-nitrophenylphosphate.

Storage Buffer
The enzyme is supplied in: 20 mM Tris-HCl (pH 8.0), 1 mM MgCl₂, 50 mM KCl, 0.1 mM ZnCl₂, 50% (v/v) glycerol.

10X Reaction Buffer
0.1 M Tris-HCl (pH 7.5 at 37°C), 0.1 M MgCl₂.

Inhibition and Inactivation

• Inhibitors: metal chelators, inorganic phosphate and phosphate analogs.
• Inactivated by heating at 85°C for 15 min or at 75°C for 10 min in the presence of 5 mM EDTA (pH 8.0), or by phenol/chloroform extraction.

Note
The Calf Intestine Alkaline Phosphatase binding to DNA may result in a band shift in agarose gels. To avoid this, prior to electrophoresis we recommend to incubate the samples with Fermentas 6X Loading Dye & SDS Solution at 65°C for 10 min and chill on ice.

Recommendations for Dephosphorylation of Proteins

Reaction Mixture: 1X reaction buffer for CIAP, 0.1-0.2 mg/ml of phosphoprotein, 10 u of Calf Intestine Alkaline Phosphatase. Incubate at 37°C for 1 hour. Note The reaction can be stopped by addition of EDTA to final 50 mM concentration or by addition of sodium orthovanadate (Na3VO4) to final 10 mM concentration. The optimal incubation time and the enzyme concentration must be determined experimentally for each substrate.

 

Related Products

T4 Polynucleotide Kinase Exonuclease I, E.coli T4 DNA Ligase T4 RNA Ligase Rapid DNA Ligation Kit Rapid DNA Ligation & Transformation Kit 6X Loading Dye & SDS Solution Water, nuclease-free

References

1. Ausubel, F.M., et al., Current Protocols in Molecular Biology, vol. 1, John Wiley & Sons, Inc., Brooklyn, New York, 3.10.1-3.10.2, 1994-2005.
2. Werle, E., et al., Convenient single-step, one tube purification of PCR products for direct sequencing, Nucleic Acids Res., 22, 4354-4355, 1994.
3. Ahmad, Z., Huang, K.P., Dephosphorylation of rabbit skeletal muscle glycogen synthase (phosphorylated by cyclic AMP-independent synthase kinase 1) by phosphatases, J. Biol. Chem., 256, 757-760, 1981.

Updated kovo 18, 2008 09:01