Pyrene Fluorescence Assay

Pyrene Fluorescence Assay of Actin Assembly

Dyche Mullins, 9/13/99

Stock solutions:

Buffer A (a.k.a. G buffer):
2 mM Tris HCl, pH 8.0
0.2 mM ATP
0.1 mM CaCl2
0.5 mM DTT

KMEI (Polymerization buffer):
50 mM KCl
1 mM MgCl2
1 mM EGTA
10 mM Imidazole HCl, pH 7.0

ME (Mg-exchange buffer):
50 µM MgCl2
0.2 mM EGTA

Useful facts:

Extinction coeff of actin at 290 nm: 38.5µM/O.D.
Correction for pyrene actin – subtract 0.127 A344 from A290
Extinction coeff of pyrene at 344 nm: 45.0µM/O.D.

PREFERRED WAVELENGTHS (low light scattering):
Excitation: 365 nm
Emission: 407 nm

ACCEPTABLE WAVELENGTHS (higher signal):
Excitation: 365 nm
Emission: 385 nm

Before starting:

1. Be sure you have good reagents.
a. Actin