Abstract
There continue to be many new technical developments that propel advances in our understanding of biological events taking place at the cellular and molecular level. One method that developed early in the expansion of cell and molecular biology and still remains a valuable tool and major core laboratory protocol is immunoblotting. Immunoblot detection of proteins, originally and often called Western blotting, is a method that takes advantage of antibodies that are raised in animals following immunization with the target protein (immunogen). The ability of the generated antibodies to recognize the target protein with high specificity and bind with high affinity makes the method very powerful. When a cell- or tissue-derived mixture of proteins containing the target is first separated based on size or charge and then immobilized on an inert support, the method becomes very informative. This chapter presents aspects of the immunoblotting technique relevant to preparations of brain vascular proteins, standard blotting protocols for optimal detection and performance, and generation of chicken-raised polyclonal antibodies.
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References
Goldstein, G W., Wolinsky, J. S., Csejtey, J., and Diamond, I. (1975) Isolation of metabolically active capillaries from rat brain. J. Neurochem. 25, 715ā717.
Gerhart, D. Z., Broderius, M. A., and Drewes, L. R. (1988) Cultured human and canine endothelial cells from brain microvessels. Brain Res. Bull. 21, 785ā793.
Harlow, E., and Lane, D. (1988) Antibodies, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 53ā239.
Gerhart, D. Z., Enerson, B. E., Leino, R. L., Zhdankina, O., and Drewes, L. R. (1997) Expression of monocarboxylate transporter MCT1 by brain endothelium and glia in adult and suckling rats. Am. J. Phys. 273, E207āE213.
Peterson, S. (1998) One-dimensional SDS gel electrophoresis of proteins. In Current Protocols in Molecular Biology, Vol. 2. Ausubel, F. M., Brent, R., Kingston, R. E., et al., eds. John Wiley and Sons, New York, unit 10.2.
Bittner, M., Kupferer, P., and Morris, C. F. (1980) Electrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzyloxymethyl cellulose or nitrocellulose sheets. Anal. Biochem. 102, 459ā471.
Harper, D. R., and Murphy, G. (1991) Nonuniform variation in band pattern with luminal/horseradish peroxidase western blotting. Anal. Biochem. 192, 59ā63.
Duelli, R., Enerson, B. E., Gerhart, D. Z., and Drewes, L. R. (2001) Expression of large amino acid transporter LAT1 in rat brain endothelium. J. Cereb. Blood Flow Metab. 20, 1557ā1562.
Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 82ā83.
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Matson, C.T., Drewes, L.R. (2003). Immunoblot Detection of Brain Vascular Proteins. In: Nag, S. (eds) The Blood-Brain Barrier. Methods in Molecular Medicineā¢, vol 89. Humana Press. https://doi.org/10.1385/1-59259-419-0:479
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DOI: https://doi.org/10.1385/1-59259-419-0:479
Publisher Name: Humana Press
Print ISBN: 978-1-58829-073-1
Online ISBN: 978-1-59259-419-1
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