Thin-layer agarose isoelectric focusing of alkaline phosphatase isoenzymes from human neutrophils

An improved method for thin-layer agarose isoelectric focusing of alkaline phosphatases (AP) from human neutrophils is described. The solubilization of AP isoenzymes was studied with four detergents. The best results were obtained after sonication with Zwittergent 3-12 (1% final concentration), followed by butanol extraction and ultracentrifugation 105,000 x g for 1 h. The cytosol can be stored at 0 degrees C or -80 degrees C, but not at -20 degrees C. Dialysis of the cytosol against a Tris buffer, pH 7.5, was imperative prior to focusing for removal of the detergent. Enzyme visualization is enhanced by incorporation of ZnCl2 (3 mM) into the agarose gel. The focusing patterns consist of two sets of isoenzymes: two main zones with pI 6.4 and 6.8 and minor components with pI 4.2, 4.8 and 5.2.