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Thin‐Layer agarose isoelectric focusing of alkaline phosphatase isoenzymes from human neutrophils
Author(s) -
BrissonLougarre Andrée,
Vergnes Henri,
Grozdea Jean,
Biermé Robert
Publication year - 1988
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150090505
Subject(s) - isoelectric focusing , agarose , isozyme , alkaline phosphatase , chemistry , thin layer , isoelectric point , biochemistry , electrophoresis , chromatography , layer (electronics) , microbiology and biotechnology , enzyme , biology , organic chemistry
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 × g for 1 h. The cytosol can be stored at O °C or −80 °C, but not at −20 °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 Zn Cl 2 (3 mM) into the agarose gel. The focusing patterns consist of two sets of isoenzymes: two main zones with p I 6.4 and 6.8 and minor components with p I 4.2, 4.8 and 5.2.