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 gel 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 1μg 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.
Human globin chain separation by isoelectric focusing in ultrathin polyacrylamide gels / K. Valkonen, E. Gianazza, P.G. Righetti. - In: CLINICA CHIMICA ACTA. - ISSN 0009-8981. - 107:3(1980), pp. 223-229.
Human globin chain separation by isoelectric focusing in ultrathin polyacrylamide gels
E. GianazzaSecondo
;
1980
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 gel 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 1μg 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.Pubblicazioni consigliate
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