A SIMPLE METHOD FOR THE DETERMINATION OF HEMOGLOBIN A2 BY MEANS OF ISOELECTRIC FOCUSING ON POLYACRYLAMIDE GEL

Quantitative determination of hemoglobin A2 and hemoglobin F is one of the important tests for the diagnosis of the β thalassemia syndrome. Generally, estimation of hemoglobin A2 have been carried out by cellulose acetate membrane electrophoresis1) and micro-column chromatography using DEAEcellulose (DE-52)2,3). In this paper an successful use of isoelectric focusing on pH gradient 6-9 of polyacrylamide gel for hemoglobin A2 assay will be described. The pH gradient 6-9 of polyacrylamide gel was prepared as follows : The mixture of acrylamide-Bis solution (3.0ml), which was prepared by dissolving acrylamide monomer (30g) and N,N'-methylenebisacrylamide (0.8g) in water and made to 100ml of final volume. To this solution were added 20.8 percent aqueous solution of sucrose (11ml), ampholine pH 3.5-10 (0.15ml), ampholine pH 7-9 (0.60ml) and water (3.0ml), mixed completely and deaerated, followed by addition of 0.004 percent aqueous riboflavin solution (0.1ml), N,N,N',N'-tetramethylethylenediamine (0.05ml) and 10 percent aqueous ammonium persulfate solution (0.05ml). The mixture thus prepared was introduced into the narrow space (1mm in thickness) which is formed between the two sheets of glass plate (130mm×180mm).