Thin-Layer Agarose Isoelectric Focusing: an Improved Technique for Determining Sheep Hemoglobin Type

Abstract

An improved technique for rapid screening of sheep flocks for hemoglobin (Hb) type is presented. This technique, isoelectric focusing (IEF) on thin-layer agarose gels is simple, rapid, inexpensive and is suitable for screening large numbers of sheep for Hb type. With this technique, up to 100 sheep blood samples can be prepared, tested and interpreted within 2 h after samples are drawn. The new technique was shown to provide better resolution than polyacrylamide gel electrophoresis (PAGE) and was able to resolve samples in which the Hb had become partially degraded. These same samples could not be resolved by PAGE. The use of a special electroendosmosis-free grade of agarose provided resolution essentially equal to polyacrylamide as a matrix for IEF. The advantages are that the casting of the agarose gels is considerably easier, the focusing of samples is more rapid, staining and destaining times are greatly reduced and hazards from potential neurotoxicity of acrylamide are eliminated. Blood from 138 ewes at the Oregon State University Sheep Center was examined by the new agarose IEF technique to determine and demonstrate its usefulness for screening. No difficulty was encountered with interpretation of any of the samples. Frequencies of the HbA and B alleles were similar to those found in earlier studies when polyacrylamide tube gel electrophoresis was used. The observed frequencies were also similar to those expected with the population in Hardy-Weinberg equilibrium.