Isoelectric focusing of hemoglobins on thin-layer agarose

Isoelectric focusing on thin layers of agarose was used to separate several human hemoglobin variants in narrow pH range (pH 6-9). Problems with gel flooding and distortions due to electroendosmotic flow were solved by altering the casting and processing of the gel, by modifying the focusing apparatus, and by utilizing commercial agarose that had been chemically modified to reduce electroendomosis. Hemoglobins C, O-ARAB, A2, and E were distinguished from one another, as were hemoglobins S, D-LOS ANGELES, G-PHILADELPHIA, F. A, I, and J. The technic is rapid, simple, and relatively inexpensive. The agarose is nontoxic, has excellent gelling properties, and possesses large pores, yet gives resolution equivalent or superior to that obtained on thin layers of polyacrylamide gel, making it preferable to polyacrylamide for thin-layer isoelectric focusing.