Resolution of Alkaline Phosphatase Isoenzymes in Serum by Isoelectric Focusing in Immobilized pH Gradients

This new methodfor fractionationof serumalkalinephospha-tase isoenzymesis basedon isoelectricfocusingon a mixed-type polyacrylamidesupportcontainingan immobilizedpH gradientwith a superimposedcarrier-ampholytegradient.All known forms of alkaline phosphatase are separated in an ImmobilinepH 3.5-6.0 gradient, the sample being applied Into pockets cast on a pH 8.0 plateau. Sharp zymogram bandsare obtained by substitutingalkaline-stable5-bromo-4-chloro-3-indoxylphosphate and tetrazollum salts for the standard1- and 2-naphthylphosphate-diazoniumsalt com-binations.After hydrolysisof the phosphate group by the alkalinephosphatasethe indoxylmoietiesreduce tetrazolium salts to nearly insolubleand nondiftusibleformazan precipi-tates. Normal sera show an array of about 10 isobands isoelectricbetween pH 3.9 and pH 4.79. In Paget’sdisease