INTRODUCTION
The use of
fluorescently labeled proteins as probes of the structure and function of
living cells began with components of the cytoskeleton. Although the great majority of
fluorescent protein analogs are now fluorescent fusion proteins, flurophore labeled proteins remain the most direct and
versatile method for studying cytoskeletal func=
tion.
Chemical synthesis of cytoskeletal probes requi=
res
the isolation of significant quantities of the target protein. Primary components of the
cytoskeleton such as tubulin, actin
and myosin are abundant and relatively easily purified in hundreds of milli=
gram
quantities from animal tissues. Fluorophores possessing amine- or thiol
-reactive groups are used to selectively modify lysine or cysteine
residues, respectively. It is essential that derivatiz=
ation
of the protein does not result in loss of function. Methods for selecting
active protein and removal of unconjugated fluorophore are usually employed subsequent to derivatization.
The choice of fluorophore at any given
wavelength of fluorescence emission should optimize fluorescence intensity =
and photostability.
We
present protocols for purification and fluorescent conjugation of brain tubulin, skeletal muscle actin
and smooth muscle myosin. All=
of
these have been used extensively and productively in our laboratory.