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High performance purification of glycolytic enzymes and creatine kinase from chicken breast muscle and preparation of their specific immunological probes



High performance purification of glycolytic enzymes and creatine kinase from chicken breast muscle and preparation of their specific immunological probes



Preparative Biochemistry 17(2): 157-172



We developed a novel procedure for isolation of the muscle isozymes of aldolase, triose phosphate isomerase (TPI), glyceraldehyde phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGM), enolase, pyruvate kinase (PK) and lactic dehdyrogenase (LDH), and also creatine kinase (CK), at high purity, specific activity and yield. Protein was extracted from chicken breast muscle and glycolytic enzymes were purified by a three step procedure consisting of: 1. Ammonium sulfate combined with pH fractionation. 2. Phosphocellulose chromatography with performance of high pressure liquid chromatography, exploiting a pH gradient formed by a gradient of the buffering ion for protein elution 3. Affinity chromatography causing elution by substrate or pH. The enzymes, obtained at over 95% purity as judged by specific activity and silver stained electropherograms, were injected into sheep. Antibody for each enzyme was purified on specific immunosorbant and it specificity was verified by immunotransfer analysis.

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Accession: 005571955

Download citation: RISBibTeXText

PMID: 3037510

DOI: 10.1080/00327488708062486



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