Purification of L-glutamate decarboxylase from rabbit brain and preparation of a monospecific antiserum.
L-Glutamate decarboxylase (GAD), the enzyme responsible for the biosynthesis of gamma-aminobutyric acid, has been purified from rabbit brain and used for the production of a monospecific rabbit antiserum. The enzyme was purified approximately 500-fold by a combination of ion exchange, hydrophobic, hydroxyapatite, and gel filtration column chromatography, and preparative polyacrylamide gel electrophoresis. The preparation thus obtained was injected into a rabbit, producing a polyspecific antiserum. This first antiserum was used for the preparation of an immunoprecipitate containing GAD; injections of the immunoprecipitate into a second rabbit led to the production of a second, monospecific anti-GAD serum. The specificity of the antiserum for GAD has been demonstrated by enzyme precipitation, immunodiffusion, line immunoelectrophoresis, gel filtration chromatography of GAD/anti-GAD immune complexes, and immunocytochemistry in the rat cerebellum.
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D000818:Animals; D001921:Brain / Q000201:enzymology*; D002845:Chromatography / Q000379:methods; D003429:Cross Reactions; D000939:Epitopes; D005968:Glutamate Decarboxylase / Q000302:isolation & purification*; D006054:Goldfish; D007106:Immune Sera / Q000276:immunology*; D007114:Immunization; D011817:Rabbits
D000939:Epitopes; D007106:Immune Sera; D005968:Glutamate Decarboxylase