Immunoaffinity purification of type I protein kinase C.
We designed a simple procedure for the purification of type I protein kinase C, using immunoaffinity chromatography with a monoclonal antibody, MC-1b, obtained by rescreening hybridoma cells available for an affinity ligand. Western blotting demonstrated that MC-1b specifically reacted with type I protein kinase C, and the enzyme molecule dissociated from MC-1b-coupled Sepharose 4B with mild eluants such as thiocyanate retained the kinase activity. A 1148-fold purification was achieved and 210 micrograms of type I protein kinase C was obtained from three rabbit brains, by means of a two-step procedure, using DEAE-cellulose and immunoaffinity chromatography. The resultant preparation was homogeneous, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis hydroxylapatite chromatography, and immunological analysis using MC-1a, MC-2a, and MC-3a.
Department of Pharmacology, Nagoya University School of Medicine, Japan.
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Hachiya
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Hagiwara
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Hidaka
D000818:Animals; D000911:Antibodies, Monoclonal; D007163:Immunosorbent Techniques; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification*; D011493:Protein Kinase C / Q000276:immunology / Q000302:isolation & purification*; D011817:Rabbits
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D011493:Protein Kinase C