Direct tissue isoelectric focusing on mini ultrathin polyacrylamide gels followed by subsequent western blotting, enzyme detection, and lectin labeling as a tool for enzyme characterization in histochemistry.
Application of cryostat sections directly onto mini ultrathin polyacrylamide isoelectric focusing gels allows an elution of proteins out of the sections into the gels under conventional focusing conditions. Protein bands representing alkaline phosphatases can easily be identified on nitrocellulose after performing a modified Western blot procedure. Furthermore, carbohydrate residues of several isoforms of alkaline phosphatases separated by isoelectric focusing can be demonstrated in a single blot strip by subsequent incubation of this strip with substrates for alkaline phosphatase and peroxidase, the latter being employed as the enzyme to which the applied lectins are covalently linked. This simple and reproducible procedure is likely to enable histochemists to determine isoforms of enzymes from a single cryostat section.
Schumacher
U
U
University of Southampton, United Kingdom.
Trudrung
P
P
D000469:Alkaline Phosphatase / Q000032:analysis*; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006651:Histocytochemistry; D007421:Intestine, Small / Q000737:chemistry / Q000166:cytology; D007525:Isoelectric Focusing / Q000379:methods; D007527:Isoenzymes / Q000032:analysis; D037102:Lectins; D009195:Peroxidase / Q000378:metabolism; D051381:Rats
D007527:Isoenzymes; D037102:Lectins; D009195:Peroxidase; D000469:Alkaline Phosphatase