Description
General description
Alkaline phosphatase (ALP) is a glycoprotein that is expressed ubiquitously. This enzyme is bound to the cell membrane. Activation of ALP requires essential co-factors, such as zinc and magnesium. Alkaline phosphatase catalyzes the removal of phosphate groups from various compounds that are phosphorylated. Alkaline phosphatase from calf intestine hydrolyzes 5′-monophosphate groups from both DNA and RNA. It can also hydrolyze 5′-diphosphate and 5′-triphosphate groups from RNA. Hence, intestinal ALP is one of the commonly used enzymes in molecular cloning.
Application
Use this preparation of calf intestinal alkaline phosphatase to remove 5′-terminal phosphates from DNA or RNA and immunoprecipitated RNA samples.
Note: The 11097075001 preparation has a high concentration of enzyme (20 U/μl), which is convenient for large-scale experiments. The 10713023001 preparation is a lower concentration (1 U/μl), which is convenient for small-scale experiments.
Physical form
10713023001: Enzyme solution (1 U/μl) in storage buffer, pH 7.6 (20 °C)
11097075001: Enzyme solution (20 U/μl) in storage buffer, pH 7.6 (20 °C)
Preparation Note
Working solution: Storage buffer: 25 mM Tris-HCl, 1 mM MgCl2, 0.1 mM ZnCl2, glycerol 50% (v/v), pH 7.6 (4 °C).
Storage conditions (working solution): Dilutions (1 to 5 U/μl) of MB grade AP in the storage buffer are stable for 17 months at 2 to 8 °C.
Higher Dilutions (~0.01 U/μl) should be made fresh daily and kept at 2 to 8 °C.
Preventing self-ligation of vectors during cloning
If a linearized vector lacks a 5′ phosphate, it cannot ligate to itself or form concatamers that contain multiple copies of the vector. Thus, the product of the ligation reaction is predominantly recombinant DNA (vector + DNA insert), rather than religated plasmid.
Inactivation: Add 1/10 volume of EGTA to the reaction mix and incubate at +65°C for 10 minutes. To ensure complete inactivation of enzyme, extract the mix with phenol/chloroform/isoamyl alcohol to remove all protein.
Other Notes
AP is a zinc-containing enzyme with 4 atoms zinc per molecule (Efstradiatis, 1977).
For general laboratory use.
One unit of alkaline phosphatase is the enzyme activity which hydrolyzes 1 μmol of 4-nitrophenyl phosphate in 1 minute at +37 °C under assay conditions.
Note: According to Moessner et al. 5 units alkaline phosphatase (+37 °C; diethanolamine buffer) correspond to 1 unit alkaline phosphatase (+25 °C; glycine/NaOH buffer).
Unit Conversion: Approx. 3.6 U (MB grade AP)
[+37 °C, 4-NPP as substrate, diethanolamine as buffer, pH 9.8] = 1 U [+25 °C, 4.NPP as substrate, glycine as buffer, pH 10.5].
Volume Activity: 20 U/μl for the 11097075001 preparation. 1 U/μl for the 10713023001 preparation.
