Human globin chain separation by isoelectric focusing in ultrathin polyacrylamide gels

Abstract

We describe three basic modifications of our previous method for thalassemia screening by isoelectric focusing of heme-free globin chains: the gel thickness is reduced from 2 mm to 240 μm; the level of the detergent Nonidet P-40 in the gels is decreased from 3% to 0.5%; the polyacrylamide slab is covalently fixed to the supporting glass plate by treatment with silane A-174. By the present method the entire focusing process, starting from gel moulding up to gel destaining and drying, is completed within 4 h, a fraction of the time needed in our previous technique. More than a 100 samples can be analyzed per working day. The present technique also affords increased sensitivity: less than l μg protein/band is detected and less than 200 picomol of hemoglobin are needed for each analysis. Band sharpness and resolution in our ultrathin gels is also considerably increased.