Calf Intestinal Alkaline Phosphatase (CIAP, CIP)
(Orthophosphoric-monoester phosphohydrolase E.C.3.1.3.1.)
Source:
Bovine intestinal mucosa
Description:
Calf Intestinal Alkaline Phosphatase (CIAP or CIP) catalyzes the hydrolysis of many phosphomonoesters, including 5'-nucleotides, RNA and DNA. It is widely used for the dephosphorylation of 5'-phosphorylated ends of DNA or RNA for subsequent labeling with 32P using [γ-32P] ATP and T4 Polynucleotide Kinase (PN 70031). Dephosphorylation also prevents religation of linearized plasmid DNA in cloning experiments. CIAP can be inactivated by phenol:chloroform extraction, by gel purification or by spin column.
Properties:
Activators: Zn2+, Mg2+, Ca2+
Isoelectric Point: 5.7
Molecular Weight: Dimer of approximately 110 kDa (two 55 kDa subunits)
Requirement for Stability: Triton X-100, zinc acetate or zinc chloride, cysteine
Optimum pH: 9 - 10
Optimum Temperature: 37°C.
pH Stability: pH 7.5; 5.0 in the presence of Triton X-100, Zn2+, cysteine
Purity:
Tested for contaminating non-specific endonucleases, exonucleases and ribonucleases.
Storage Buffer:
10mM Tris-HCl (pH 7.5), 5mM MgCl2, 0.1mM ZnCl2, 50% glycerol.
Assay Conditions:
The reaction mixture (1 ml) contains 100mM glycine-HCl (pH 9.6), 1mM MgCl2, 1mM
p-nitrophenol phosphate and 0.01 - 0.1 units of enzyme at 37°C. The change in absorbance
at 410 nm is monitored.
Unit Definition:
One unit is the amount of enzyme required to catalyze the hydrolysis of 1 μmol of
p-nitrophenyl phosphate per min at pH 9.6 (glycine/NaOH buffer) and 25°C. Note that this is equivalent to 5 units of enzyme assayed at 37°C in diethanolamine buffer (pH 9.8) as reported by some other suppliers.
Concentration:
20 units/μl
Tested User Friendly™ Functional Test:
Dephosphorylation of a restriction enzyme digested plasmid (5 - 10 pmol of 5' ends, 0.01 - 0.05 units/pmol 5' ends. Reduces re-ligation to <0.5% compared to the untreated control.)
Functionally Tested 10X CIAP Reaction Buffer (1 ml included):
200mM Tris-HCl (pH 8.0), 100mM MgCl2.
CIAP Dilution Buffer (1 ml included, PN 72765):
50mM Tris-HCl (pH 8.0).
References:
- ZHANG, Y., ZHU, Y. AND ZHOU, H. (2000) Int. J. Biochem. Cell. Biol. 32, 887-894.
- ABDOLRAZAGHI, Z. AND BUTTERWORTH, P. (1983) Enzyme 30, 12-20.
- BAUNOCH, D. A., et al. (1992) Oncogene, 7, 2351-2353.
Applications:
- Dephosphorylation of 5' termini of nucleic acids before labeling with T4 Polynucleotide Kinase.
- Dephosphorylation of 5' termini of DNA fragments to prevent self-ligation.
Storage:
Shipped on dry ice. Store at -20°C.