Purification of skeletal muscle actin
The isolation of muscle actin (G. Feuer et al., 1=
948) is
one of the most time-honored protein purification procedures with only minor
modifications to the protocol over 5 decades of use. The purification depends on the
differential salt solubility of actin and myosi=
n that
together comprise nearly 80% of the protein of the contractile apparatus. Myosin is extracted from minced mu=
scle
with high salt. After extensive washing with cold alkaline water, the resid=
ue
is dehydrated with acetone to remove lipids and denature nonactin
proteins. G-actin
is extracted with low salt and then converted to polymeric F-actin by increasing the salt concentration. Tropomyosin is
removed by treating the F-actin with high salt =
and
the polymerized actin collected by
ultracentrifugation.
Prepa=
ration
of rabbit muscle acetone powder
&n=
bsp;
1.&n=
bsp;
This
procedure is for 100 g of rabbit muscle. The muscle (dorsal lateral skeletal
muscle and hind leg muscle) should be excised immediately after sacrifice a=
nd chilled
on ice for present use or sealed securely in plastic wrap and frozen at
–20 or –80o>. Frozen muscle is good for at least a
year.
2.&n=
bsp;
Mince
the muscle using a prechilled meat grinder in a=
cold
room.
3.&n=
bsp;
Extract
the mince using 3 volumes (300 ml) of ice cold Extraction Buffer (0.5 M KCl,
0.1 M K2HPO4) with stirring (an overhead paddle stirr=
er
is best, but a large magnetic stir bar will do) on ice for 10 minutes.=
4.&n=
bsp;
Distribute
the slurry to 500 ml bottles and centrifuge at 3000 rpm in a Sorvall GS-3 rotor at 4o for 15 minutes. D=
iscard
the supernatant.
5.&n=
bsp;
Re-extract
the pellet with the same volume of Extraction for 10 minutes on ice. Stirri=
ng
can be done in the centrifuge bottle.
6.&n=
bsp;
Repeat
the centrifugation. Discard the supernatant.
7.&n=
bsp;
Add
3 ml of ice-cold distilled water per gram of original muscle (300 ml) to the
pellet. Stir in the centrifuge bottle using a glass rod.<=
/p>
8.&n=
bsp;
Adjust
the pH to 8.2-8.5 with 1 M sodium carbonate.
9.&n=
bsp;
Centrifuge
at 3000 rpm for 10 minutes at 4o. Discard supernatant and mark t=
he
volume of the pellet on the bottle (alternatively the bottle can be weighed=
).
10.&=
nbsp;
Repeat
Steps 7- 9 about 4 times. Check the pH and maintain it at 8.2-8.5. As soon as the pellet begins to sw=
ell
(or the weight increases) continue to the next step regardless of the numbe=
r of
washes carried out. Do not over wash!
11.&=
nbsp;
Add
2-3 volumes of cold acetone (on ice) to the pellet and stir vigorously (usi=
ng a
large magnetic stir bar) for 20 minutes in a hood. CAUTION: acetone is high=
ly
flammable!
12.&=
nbsp;
Centrifuge
at 3000 rpm for 10 minutes at 4o . Discard the acetone-containing
supernatant using an approved method of waste disposal.
13.&=
nbsp;
Repeat
Steps 11 and 12.
14.&=
nbsp;
Spread
the acetone precipitate on a large piece of filter paper in a hood and dry =
at
room temperature overnight.
15.&=
nbsp;
Store
the powder in 10 g aliquots desiccated at –20o . It is stable for at least 6 months=
.
Solutions:
Extraction Buffer: 600 ml =3D 44.8 g KCl,
10.4 g K2HPO4
1 M Na2CO3: 100
ml =3D 10.6 g