Tetramethylbenzidine staining procedure after starch gel electrophoresis of human haemoglobin

The purpose of the present work was to increase the stability of tetramethylbenzidine-stained electrophoretic patterns of human haemoglobin through use of a suitable fixing agent. Since haemoglobin examination is quite important in the study of numerous pathologies, in prenatal analyses and in forensic medicine, it was considered worthwhile to attempt to stabilise the specific tetramethylbenzidine-based stain, which is notoriously unstable. The other stain frequently used is amido black 10 B, a generic protein stain, which reveals both haemoglobin and non-haemoglobin bands, hence interpretation of the results can lead to errors and the identification of false pattern heterogeneity. Following several experimental tests we have observed that it is possible to make tetramethylbenzidine-based stains stable by using a suitable fixative. Treatment with the fixing solution prevents the colour from fading, and can keep it intact for over a year. The method also appears promising for studying haemoglobin in a variety of other species of vertebrates.