Purification of Bovine Carbonic Anhydrase by Affinity Chromatography: An Undergraduate Biochemistry Laboratory Experiment

Autor:	C. Larry Bering, Jennifer J. Kuhns, Roger S. Rowlett
Rok vydání:	1998
Předmět:	Lysis Chromatography biology Chemistry Elution General Chemistry Ligand (biochemistry) Education Electrophoresis chemistry.chemical_compound Biochemistry Affinity chromatography Carbonic anhydrase biology.protein Sodium dodecyl sulfate Polyacrylamide gel electrophoresis
Zdroj:	Journal of Chemical Education. 75:1021
ISSN:	1938-1328 0021-9584
DOI:	10.1021/ed075p1021
Popis:	We have developed a rapid and inexpensive experiment utilizing affinity chromatography to isolate carbonic anhydrase (CA) from bovine blood. The more specific an affinity gel is the better the purification, but the greater the cost. Some costs would be prohibitive in the undergraduate biochemistry laboratory. Less specific resins may be more affordable but may bind a number of closely related proteins. One alternative would be to couple a specific ligand to an inexpensive resin such as an ion exchanger. We describe a simple procedure for preparing a sulfonamide-coupled resin which specifically binds CA from a blood hemolysate. The CA is eluted and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It was found that only a single band of 31 kD was obtained. The instructor can readily prepare the affinity gel prior to the lab, and the students, beginning with packed red blood cells can carry out the lysis, binding to the gel, elution, enzymatic assays, and electrophoresis.
Databáze:	OpenAIRE
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