CIAP-Calf Intestinal Alkaline phosphotase.
Description
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.
Unit definition

One unit of the enzyme hydrolizes 1µmol of 4-mitrophenylphosphate to 4-nitrophenol in 1 min at 37°C.

Reaction buffer
#B22 (x10): 100 mM Tris-HCl pH 8.3, 10 mM MgCl2, , 10 mM ZnCl2, .
Reaction conditions
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).
Storage
Supplied in storage buffer without glycerol. Store at +4°C.
Applications
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.

Quality control

activity in self-ligation test, SDS-PAGE purity, absence of contaminating nucleases.

References

1. Engstrom, L. (1961) Biochim. Biophys. Acta 52, 36-48. 2. Mšssner et al.. (1980) Z. Physiol. Chem. 361, 543-549.

Catalog #
M9.4

 

 

Pack size

1000 U

 

 

 

Copyright © 2002 M.B. Enzymes GmbH