Download PDF by John J. Correia, Leslie Wilson: Microtubules, in vitro

By John J. Correia, Leslie Wilson

ISBN-10: 0123748151

ISBN-13: 9780123748157

There remains to be excessive curiosity within the microtubule cytoskeleton, the meeting, constitution and law of microtubules and the varied cars and accent proteins that keep an eye on mobile cycle, dynamics, association and delivery. the sector maintains to develop and discover new facets of those concerns pushed immensely through advancements in optical imaging and monitoring suggestions. This volume (complimented by means of the approaching better half quantity via Cassimeris and Tran) brings jointly present learn and protocols within the box of microtoublues in vitro and will serve as a valuable tool for cell biologists, biophysicists and pharmacologists who research the microtubule cyctoskeleton, in addition to for researchers within the biomedical and biotechnology groups with curiosity in constructing medicinal drugs that focus on microtubules, MAPS and cars. * Chapters replicate both experimental strategies and new advancements within the box of Microtubule in vitro research.* Combines classical methods and smooth applied sciences* provides easy-to-use protocols and thorough history info, compiled via leaders in the sphere

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The characterization of AA2 exemplifies the procedures that we have employed to characterize isotype-specific tubulin antibodies as well as other proteins. , 1984). Mouse IgGs are purified from tissue culture supernatants by conventional Protein-G chromatography. Rabbit IgGs are purified first from serum proteins by Protein-A chromatography, and specific IgGs are then isolated by peptide affinity chromatography. The mouse monoclonal antibodies and the affinity purified rabbit polyclonal antibodies are screened first by enzyme-linked immunosorbent assay (ELISA) to determine their reaction with nondenatured purified bovine or rat brain tubulin.

B. Genetic Manipulation of Tubulin Genes in T. thermophila One of the original biochemical sources for tubulin is the free-living protozoan T. thermophila; tubulin was first purified from Tetrahymena in 1976 (Kuriyama, 1976). Tetrahymena is a ciliate, with an abundant microtubule cytoskeleton. , 1993). Due to its unusual nuclear organization and life cycle, it is possible to knock out essential genes in Tetrahymena and to later select for genes to complement this loss. Tetrahymena contains two nuclei: a “germ line” micronucleus which is a transcriptionally silent “backup” copy of the genome and a “somatic” macronucleus which contains the actively transcribed genome.

J. Parasitol. 22, 939–946. Gupta, M. , Bode, C. , Georg, G. , and Himes, R. H. (2003). Understanding tubulin-Taxol interactions: Mutations that impart Taxol binding to yeast tubulin. Proc. Natl. Acad. Sci. A. 100, 6394–6397. Gupta, M. , Bode, C. , Thrower, D. , Pearson, C. , Suprenant, K. , Bloom, K. H. (2002). beta-Tubulin C354 mutations that severely decrease microtubule dynamics do not prevent nuclear migration in yeast. Mol. Biol. Cell 13, 2919–2932. , and Gorovsky, M. A. (1997). Germ-line knockout heterokaryons of an essential alpha-tubulin gene enable high-frequency gene replacement and a test of gene transfer from somatic to germ-line nuclei in Tetrahymena thermophila.

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Microtubules, in vitro by John J. Correia, Leslie Wilson

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