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1998
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In the Laboratory
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Purification of Bovine Carbonic Anhydrase by Affinity Chromatography: An Undergraduate Biochemistry Laboratory Experiment
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C. Larry Bering and Jennifer J. Kuhns
Department of Chemistry, Clarion University, 840 Wood St., Clarion, PA 16214
Roger Rowlett
Department of Chemistry, Colgate University, 13 Oak Drive, Hamilton, NY 13346
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August 1998
Vol. 75 No. 8
p. 1021
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| Abstract |
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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.
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| More Information |
 Citation |
Bering, C. Larry; Kuhns, Jennifer J.; Rowlett, Roger. J. Chem. Educ. 1998 75 1021.
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Keywords |
Laboratory Instruction, Enzymes, Biotechnology, Biochemistry
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History |
Created:
Last Updated: |
June 22, 1999
June 24, 2005
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| Home > JCE Print > Journal of Chemical Education > Issues >
1998
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