Chapter 9: Tubules\Filaments - Endnotes
It is convenient to use permanent photographic slides,
since the microscope preparations are unstable (flurorescence
causes bleaching of the dye). Several companies produce
fluorescent antibodies to tubulin and also supply protocols for
producing the slides. Refer to Exercise 2.7 for further details.
Available from slaughter houses, or any animal brain
may be substituted. It is important that the brain be freshly
removed from a sacrificied animal, however.
From Shelanski, M.L., F. Gaskin and C.R. Cantor. 1973.
"Microtubule assembly in the absence of added nucleotides." PROC.
NATL. ACAD. SCI. USA 70:765-768. For a modified Weissenberg
Technique and Sephadex Purification of tubulin cf. G.C. Na and
S.N. Timasheff "Physical Properties of Purified Calf Brain
Tubulin" in Methods in Enzymology, Vol. 85, p. 393-408. 1982.
A household blender should only be used as a last resort.
Crude estimation of an increase in viscosity can be had by observing the rate of escape for trapped air bubbles as the solution is swirled. Viscosmeters are available commercially at reasonable prices. Alternatively, a pipette can be utilized. Mix a reasonable sample of sucrose concentrations (look up viscosities in handbook of physical constants) and simply calibrate the viscometer by measuring the time it takes to deliver 3 ml from the filled pipette.
Alternatively, the pellets containin microtubules can
be collected, fixed, embedded and sectioned for EM examination.
This will require several days, however, and will yield sectioned
views of the tubules, rather than intact whole tubules.
Cooke, P. "Preparation of Isolated Thick and Thin
Filaments" in Methods in Enzymology, Vol. 85, pp 277-284., 1982.
It is possible to isolate the individual fibers from
various sources, but avian gizzards have been most often used for
preparation of thick and thin filaments. They can be obtained
from chicken slaughterhouses in mass quantity, and are virtually
pure smooth muscle.
Aamodt, E.J. and R.C. Williams, Jr. 1984.
"Microtubule-associated proteins connect microtubules and
neurofilaments in vitro." Biochemistry 23:6023-6031.
In Methods in Cell Biology Vol. 27: Echinoderm
Gametes and Embryos (T.E. Schroeder, ed.). Academic Press,
Orlando, 1986. pp. 189-216.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- email@example.com