title: Purification of glucose-6-phosphate dehydrogenase from rabbit reticulocytes by immunoaffinity chromatography.
abstract: Glucose-6-phosphate dehydrogenase (G6PD) was purified from rabbit reticulocytes by using a single immunoaffinity chromatographic step. Antibodies against rabbit erythrocyte G6PD were raised in a goat, purified near to homogeneity and immobilized on CarboLink gel (Pierce). Nonspecific binding sites on the matrix were saturated with myokinase from rabbit muscle. Reticulocyte lysate was directly loaded onto the column, allowed to enter the gel bed and incubated for 15 min at room temperature. The column was washed first with PBS and then with 1 M NaCl. The enzyme was eluted with 0.1M acetic acid in 1M NaCl. Two protein bands of about 66.2 kDa were co-eluted with the G6PD, which was however well separated in SDS-PAGE. The eluent destroyed the enzyme activity but the G6PD yield was higher than 90%.
authors.affiliation: Istituto di Chimica Biologica G. Fornaini, Università di Urbino.
authors.forename: P
authors.initials: P
authors.lastname: Ninfali
authors.forename: S
authors.initials: S
authors.lastname: Marinoni
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002021:Buffers; D002846:Chromatography, Affinity / Q000379:methods*; D005954:Glucosephosphate Dehydrogenase / Q000097:blood*; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G; D007158:Immunologic Techniques; D015636:Magnesium Chloride; D009249:NADP; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*
chemical_list: D002021:Buffers; D007074:Immunoglobulin G; D015636:Magnesium Chloride; D009249:NADP; D005954:Glucosephosphate Dehydrogenase