Abstract
Western blotting provides an effective, sensitive, and specific method for the identification and quantitation of cyclooxygenase protein expression in an unknown complex protein sample. Electrophoresis is used to separate complex mixtures of proteins. Denaturing discontinuous one-dimensional gel electrophoresis separates proteins only based on molecular size as they move through a SDS-polyacrylamide gel (SDS-PAGE) toward the anode with the smaller proteins migrating faster and bigger proteins running slower. The SDS-PAGE is a separating gel topped by a stacking gel and secured in an electrophoresis apparatus. Sample proteins are solubilized by boiling in the presence of SDS and equal amounts of the protein solution are loaded into a gel lane, and the individual proteins separated electrophoretically. 2-Mercaptoethanol (2-ME) and dithiothreitol (DTT) are added to reduce disulfide bonds.
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References
Amersham. ECL Western Blotting Protocol. Amersham International Plc. Little Chalfont, Buckinghamshire, England.
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. S. (1997) Current Protocols in Molecular Biology. Wiley.
Guan, Z. H., Baier, L. D., and Morrison, A. R. (1997) P38 mitogen-activated protein kinase down-regualtes nitric oxide and up-regulates prostaglandin E2 biosynthesis stimulated by interleukin-1. J. Biol. Chem. 272, 8083–8089.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Sigma. Bicinchoninic acid protein assay (Sigma Procedure No. TPRO-562. For kit No. BCA-1 and Product No. B-9643). Sigma Chemical Company, St. Louis, MO.
Tetsuka, T., Daphna-Iken, D., Miller, B. W., Guan, Z. H., Baier, L. D., and Morrison, A. R. (1996) Nitric oxide amplifies interleukin 1-induced cyclooxygenase-2 expression in rat mesangial cells. J. Clin. Invest. 97, 2051–2056.
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© 1999 Humana Press Inc.
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Guan, Z., Morrison, A.R. (1999). Assessment of Cyclooxygenase Protein Expression by Western Blotting. In: Lianos, E.A. (eds) Eicosanoid Protocols. Methods in Molecular Biology, vol 120. Humana Press. https://doi.org/10.1385/1-59259-263-5:67
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DOI: https://doi.org/10.1385/1-59259-263-5:67
Publisher Name: Humana Press
Print ISBN: 978-0-89603-667-3
Online ISBN: 978-1-59259-263-0
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