Fluor=
escent
labeling of actin2
Actin=
is l=
abeled
as F-actin to protect assembly sites.
1.&n=
bsp;
Dialyze
2.5 ml of G-actin at 2-3 milligrams ml-1
against 200 ml of Labeling Buffer (50 mM PIPES,=
pH
6.8, 50 mM KCl, 0.1=
mM CaCl2, 0.2 mM ATP)
overnight at 4o to produce F-actin.<=
o:p>
2.&n=
bsp;
Add
with rapid mixing 5-(and 6)-carboxytetramethyl =
rhodamine succinimidyl es=
ter
(TMR-SE, Molecular Probes, Eugene, Oregon) to 0.3 mM
from a stock solution of 30 mM (16 milligrams m=
l-1)
in dry dimethyl formamide<=
/span>. The stock can be stored at –<=
span
class=3DGramE>80o =
for
years.
3.&n=
bsp;
Incubate
for 45 minutes at room temperature.
4.&n=
bsp;
Stop
the reaction by adding 50 mM lysine from a stoc=
k of 1
M lysine.
5.&n=
bsp;
Centrifuge
in a Beckman TLA 100.3 rotor at 50,000 rpm (k factor =3D 64) for 40 minutes=
at 4o. =
6.&n=
bsp;
Resuspend the labeled F-actin pellet i=
n 1.0
ml G buffer using a teflon-glass homogenizer. <=
o:p>
7.&n=
bsp;
Dialyze
overnight against 500 ml of G Buffer at 4o .=
8.&n=
bsp;
Centrifuge
in a Beckman TLA 100.3 rotor at 50,000 rpm for 40 minutes at 4o.
9.&n=
bsp;
To
eliminate free dye, apply the supernatant to a 5 ml Ex=
cellulose
desalting column (Pierce, Rock=
ford,
IL) equilibrated with Buffe=
r A.
Collect 0.5 ml fractions. The sample will separate into two distinct red ba=
nds
with the excluded TMR-actin eluting before free
dye. Pool the TMR-actin peak, adjust it to 5 mM Tris, pH=3D8.0, 1 mM DTT,=
1mM ATP
and drop-freeze 20 microliter aliqouts
in liquid nitrogen and store at –80o .
10.&=
nbsp;
To
determine the dye to protein ratio (D/P), measure the absorbance at 280 nm =
and
555 nm (absorbance maximum for TMR). The extinction coefficient at 280 nm f=
or actin is 4.9x 104 M-1cm-1<=
span
style=3D'mso-spacerun:yes'> and for TMR at 555 nm =
is 6x
104 M-1cm- 1. The contribution of TMR to absorba=
nce at
280 nm is 0.3 times its absorbance at 555 nm that must be subtracted from t=
he
absorbance at 280 nm to calculate the concentration of actin. A typical preparation has a D/P of
0.5-1.0 and a yield of 30-50%>.
Solutions:
Labeling Buffer: 200 ml =3D 3.02 g PIPES, 0.75 g KCl,
0.02 ml of 1.0 M CaCl2 stock, 0.4 ml of 0.1 M ATP stock.
1 M lysine monohydrochloride:
10 ml =3D 1.83 g
2 This protoco=
l is
modified from H. Isambert, P. Venier,
A.C. Maggs, A Fattuom, R. Kassab, D. Pantaloni and =
M.F. Carlier, J. Biol. Chem. 270: 11437-11444 (1995).