- CIAP-Calf Intestinal Alkaline phosphotase.
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- Description
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- Alkaline phosphatase (EC 3.1.3.1) purified from calf intestine is
a glycoprotein with M.W of 100-140 kDa which catalyzes the removal of
5´- or 3´-phosphate from RNA, DNA and ribo- and deoxyribonucleoside
di- or triphosphates. M.W. approx. 14000. Activity 3000 U/mg protein.
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- Unit definition
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One unit of the enzyme hydrolizes 1µmol of 4-mitrophenylphosphate
to 4-nitrophenol in 1 min at 37°C.
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- Reaction buffer
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- #B22 (x10): 100 mM Tris-HCl pH 8.3, 10 mM MgCl2, , 10 mM
ZnCl2, .
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- Reaction conditions
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- For dephosphorylation of DNA use 1 U of the enzyme for 1-20 pM DNA
termini. Incubate in 1x reaction buffer for 30-60 min at 37°C. Stop
reaction by heating at 75°C for 10 min. The DNA treatment with CIAP
can be directly performed simultaneously or after the cleavage by a
restriction endonuclease. In this case, ZnCl2 should be added
to the reaction mixture at a final conc. of 1mM. Ends concentration
for linear DNA can be approx. calculated as follows: pM ends=pM DNA
x (number of cuts x2 + 2); 1 µg of a 3 kb linear DNA contains
1 pM of 5´ termini. Dephosphorylation of DNA with flush and recessed
termini and dephosphorylation of RNA are more efficient performed at
higher temperatures (50-60°C).
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- Storage
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- Supplied in storage buffer without glycerol. Store at +4°C.
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- Applications
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- used as a reporter in detection systems for proteins and nucleic
acids detection; dephosphorylation of 5´ termini of nucleic acids
in cloning procedures to prevent self-ligation; subsequent tagging with
radiolabeled phosphate using T4 PNK.
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Quality control
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activity in self-ligation test, SDS-PAGE purity, absence of contaminating
nucleases.
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References
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1. Engstrom, L. (1961) Biochim. Biophys. Acta 52, 36-48. 2. Mssner et
al.. (1980) Z. Physiol. Chem. 361, 543-549.
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- Catalog #
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- M9.4
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Pack size
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1000 U
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