Alkaline Phosphatase (AP) Protocol

Dephosphorylation of proteins

Alkaline phosphatase (AP; Product No. API-RO) is a non-specific phosphomonoester hydrolase that catalyzes the hydrolysis of a wide variety of organic monophosphates. The widespread occurrence of AP in nature suggests its involvement in fundamental biochemical processes, however, there is no positive evidence regarding its physiological function, or the nature of the natural substrates. Hydrolysis of phosphoesters, phosphate transferase activity, protein phosphatase activity, phosphate transport, modulation of organic cation transport, and involvement in cell proliferation have been suggested as possible functions of ALP. Thus, alkaline phosphatase may potentially also hydrolyze phosphate groups of proteins, but we cannot provide any in-house data and procedures concerning dephosphorylation of proteins.

Two publications are available which describe dephosphorylation of proteins using Calf Intestinal Alkaline Phosphatase (CIAP) from Roche Applied Science:

Dephosphorylation by CIAP according to these protocols:

Reference 1: In a reaction mixture containing 50 mM Tris-Cl buffer, pH 7.5; 1 mM dithiothreitol; 5% glycerol; 5 mM MgCI₂; 0.1 to 0.2 mg/mL of phosphoprotein, and 10U alkaline phosphatase.

Reference 2: Heat stable MAPs (5 mg/mL) were treated with calf intestinal phosphatase in buffer containing 0.1 M Tris HCI, pH 8.4, 1 mM MgCl₂, 1 mM EGTA, 1 mM EDTA, 2 mM PMSF, 1 μg/mL pepstatin, 1 U/mL aprotinin, and 0.1 mM ZnCl₂ for 12 h at 37 °C. From a range of commercial preparations tested, only that from Boehringer Mannheim (Roche Applied Science Cat. No. 713 023, 25 U/mL) combined good phosphatase activity with lack of detectable proteolytic activity towards MAPs. Nevertheless, protease inhibitors were used in all experiments. To achieve maximal dephosphorylation, fresh phosphatase was added to the reaction every 3 h to a final concentration of 100 U/mL. The phosphatase was subsequently inactivated by heating for 2 min in a boiling water bath.

Note: Not all phosphorylation sites are accessible for dephosphorlyation by AP (due to steric hindrances), and other phosphatases (e.g., specific phosphoprotein phosphatases or acidic phosphatases) may have to be used.

Inactivation

Alkaline phosphatase can be inactivated by heat: Heat the reaction for 10 min at +65 °C followed by at least one extraction with phenol/chloroform/isoamylalcohol (50 : 48 : 2). AP inactivation by heat depends upon the protein concentration and the divalent ion concentration (Mg²⁺, Zn²⁺). For inactivation of AP, molecular biology-grade following a dephosphorylation reaction, heat the reaction 30 min at +65 °C in the presence of 50 mM EGTA (NOT EDTA; EGTA is needed to chelate divalent cations), then extract with phenol and precipitate the nucleic acid with ethanol.

The amount of EGTA required depends upon the Mg²⁺ concentration of the reaction mix: If the reaction mix contains no Mg²⁺ use 5 mM EGTA (final conc.); if the reaction mix contains 1 mM Mg²⁺ use 10 mM EGTA. Phenol extraction should always be included in the inactivation procedure if the AP concentration is higher than 0.3 U/10 μL, to insure that AP does not interfere with further steps. However, if time or amount of nucleic acid preclude ethanol precipitation, a longer incubation (up to 45 min) at +65 °C in the presence of EGTA is usually sufficient to completely inactivate AP. During the heat step, centrifuge the reaction mix briefly (few sec) every 10 min to collect liquid which has been condensed on the tube walls.

Enzyme concentration for dephosphorylation of DNA or RNA in reaction:

Roche recommends: - 0.5 U/50 pmol for 5′-protruding ends [30 min, +37 °C]; - 2.0 U/pmol for blunt of 5′-recessed ends [1 hour, +50 °C]; - 0.5 U/50 pmol for RNA with 5′-phosphates [1 hour, +50 °C].

Unsuccessful dephosphorylation

If a certain nucleic acid is not dephosphorylated under the above reaction conditions, try using higher Mg²⁺ concentrations and spermidine. For 5 pmol of 5′-phosphate termini, dissolve the nucleic acid in 10 μL of 50 mM Tris-HCl/pH 8.0, containing 10 mM MgCl₂, 0.5 mM spermidine, 0,01 mM EDTA and 0.1 mM ZnSO₄ at room temperature. Do not place on ice, since the spermidine-DNA complexes precipitate when cooled. Add 0.5 U AP (in 1 μL) and incubate the reaction mix at +37 °C for 30 min. Add a second aliquot of AP (0.5 U) and incubate further 30 min. To inactivate AP, heat the samples 20 min at +68 °C, add 1 μL 0.25 M EGTA (pH 7.8) and incubate a further 20 min at +68 °C. [Throughout the two heating steps, centrifuge the sample tubes a few second after every 10 min at +68 °C, to remove condensation from the tube walls and prevent precipitation of spermidine-DNA complexes.]