Comparison of diazo-coupling, formazan, and silver staining techniques for visualizing alkaline phosphatase isoenzymes after electrophoresis in homogeneous-pore and gradient-pore polyacrylamide gels.
Three techniques for visualization of alkaline phosphatase after polyacrylamide-gel electrophoresis are compared. These are diazo-dye simultaneous coupling with the substrate sodium naphthyl phosphate and 5-chloro-2-toluene diazonium chloride; formazan precipitation with the substrate 5-bromo-4-chloro-3-indolyl phosphate and 3-[4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; and silver staining with the substrate sodium glycerophosphate. Each staining technique was tested with gradient-pore and homogeneous-pore acrylamide-gel electrophoresis. The main factors assessed are sensitivity; separation of the human serum alkaline phosphatase isoenzymes of the liver, bone, and intestinal types; and differences in substrate affinity, as well as the complexity of each technique. Using the three techniques only minor differences in substrate affinity are evident. There is some nonspecific staining with the diazo-coupling technique but not with the formazan and silver staining techniques. The differences, in the mobility of the liver, bone, and intestinal isoenzymes achieved by homogeneous-pore gel electrophoresis are sufficient to allow them to be clearly distinguished. However, only very small differences in mobility are found with gradient-pore gel electrophoresis, but the sharper bands in this medium allow much smaller amounts of activity to be detected. As little as 160 microU of enzyme can be visualized by the diazo technique. Silver staining gives an approximately fourfold increase in sensitivity over the formazan technique, which in turn gives a fourfold increase over the diazo technique. An important aspect of the silver staining technique is the potential of increasing sensitivity much further by improvements in the photographic physical development stage.(ABSTRACT TRUNCATED AT 250 WORDS)
Hodson
A W
AW
Department of Clinical Biochemistry and Metabolic Medicine, Medical School, University of Newcastle upon Tyne, United Kingdom.
Skillen
A W
AW
D000469:Alkaline Phosphatase / Q000032:analysis*; D001391:Azo Compounds; D001842:Bone and Bones / Q000201:enzymology; D004396:Coloring Agents; D004591:Electrophoresis, Polyacrylamide Gel; D005562:Formazans; D006801:Humans; D007422:Intestines / Q000201:enzymology; D007527:Isoenzymes / Q000097:blood*; D008099:Liver / Q000201:enzymology; D012834:Silver; D013194:Staining and Labeling
D001391:Azo Compounds; D004396:Coloring Agents; D005562:Formazans; D007527:Isoenzymes; D012834:Silver; D000469:Alkaline Phosphatase