Purification of Skeletal Muscle Actin
Skeletal muscle is the most important preparative
source for actin since the discovery of the protein in
the 1940s. The original purification procedure developed
by Feuer et al.
(1948) is still the basis of modern
protocols. In a first step, fresh skeletal muscle tissue is
ground and most of the myosin is extracted by 0.5M
KCl. The tissue is then washed several times with distilled
water and is dehydrated with acetone at -15°C to yield acetone powder, which can be stored conveniently.
The second step, the actual actin preparation
from this acetone powder, is based on cycles of actin
extraction/depolymerization in low salt buffer, actin
polymerization by the addition of salt, and sedimentation
of the polymerized actin. Important methodical
improvements were reached by the development of
additional purification steps aimed at removing major
protein contaminants. In particular, α-actinin (Ebashi
and Maruyama, 1965) is eliminated by low-speed sedimentation
of a thick gel formed with actin at 3 M KCl,
and tropomyosin (Spudich and Watt, 1971) is dissociated
from F-actin at high ionic strength. The procedure
presented here is based on the widely used protocol of
Spudich and Watt (1971; reviewed in Pardee and
Spudich, 1982) followed by a gel filtration step at
low ionic strength to isolate monomeric ATP-G-actin
(MacLean-Fletcher and Pollard, 1980). It yields very
high purity actin suitable for all common biochemical
applications, such as actin binding and polymerization
assays or chemical labelling. To store and handle actin
efficiently, it is paramount to have an understanding
of its biochemistry, especially of its interaction with
(for a review, see Carlier et al.
At slightly alkaline pH, Ca-G-actin can be stored on
ice at 2-3mg/ml (50-75µM
) without detectable selfassembly
for weeks. This is not the case with Mg-actin,
the prevalent species in vivo
. The following protocol
includes a procedure to convert Ca-actin to Mg-actin
(Gershman et al.
II. MATERIALS AND
If not stated otherwise, chemicals are p.a. grade and
are purchased from Merck. NaaATP is from Roche
Molecular Diagnostics (0519987). Water is MilliQ
grade. Acetone should be pure (99.5%). Sephadex G-
200 is from Amersham Biosciences.
III. LABORATORY EQUIPMENT
Acetone powder preparation: sharp knife, large
glassware (4-liter beaker, 4-liter Btichner flask), inox
beaker, meat grinder, kitchen blender, gauze, and centrifuge.
Actin purification: tissue grinder, thermometer,
preparative ultracentrifuge, glass wool, dialysis
tubes, and low-pressure chromatography system.
A. Preparation of Acetone Powder from
Rabbit Skeletal Muscle
Note that acetone powder is also available commercially
(Sigma M-0637). To prevent protease activity/contamination, the material should not be
allowed to warm above 4°C and gloves should be
worn throughout the preparation.
- Extraction solution: For 8 liters, add 298.2 g KCl and
40 g KHO3 to 8 liters H2O
- Acetone: Chill to -20°C (acetone denatures proteins
- H2O: Chill to 4°C
B. Preparation of Ca-G-Actin from Acetone
- Mince preparation. If possible, work in a group
of two or three. Sacrifice two male rabbits (New
Zealand white or equivalent). Skin and place in a large
flat ice box. With a sharp knife, quickly excise the large
muscles on both sides of the spine and from both hind
legs. Chop muscles coarsely with scissors, removing as
much connective tissue as possible. Grind. You should
obtain about 600 g mince.
- Myosin extractions. Place the mince in a large
beaker and add 3 liters chilled extraction solution. Stir
for 10 min and then centrifuge for 10 min at 2500g.
Discard supernatant and repeat this step once.
- Water extractions. Discard supernatant and add
pellet to 3 liters distilled water. Stir for 10min and centrifuge
as in step 3. Repeat this step three more times
or until pellet begins to swell visibly; in this case, discontinue
- Acetone washes. Discard supernatant and mix
washed tissue with cold acetone. Homogenize briefly
with a kitchen blender. Centrifuge at 1200g for 5min.
Repeat this step two more times.
- Drying. After last acetone wash, pool all pellets
in a large inox beaker and add 2 liters cold acetone. Stir
vigorously for 20min. Lay out a large Bfichner funnel
with several layers of gauze and place on a 4-liter
Bfichner flask. Apply suction and filter the acetone
suspension. Squeeze out residual acetone by gathering
the ends of the gauze and wringing. Spread the dehydrated
material on filter paper and let dry overnight.
Store dry acetone powder at -20°C.
Gloves should be worn throughout the purification
to avoid protease contamination.
To prepare ATP-containing buffers, it is convenient
to use a stock solution of ATP in H2
O buffered to pH
7.0. Store this stock solution at -20°C.
- X buffer: 2 mM Tris-HCl, 0.5 mM ATP, 0.1 mM CaCl2,
1 mM dithiothreitol (DTT), 0.01% (w/v) NAN3, pH
7.8. Store at 4°C.
- Xb buffer: 2 mM Tris-HCl, 1 mM MgCl2, 1 mM DTT,
pH 7.8. Store at 4°C.
- G buffer: 5 mM Tris-HCl, 0.2 mM ATP, 0.1 mM CaCl2,
1 mM DTT, 0.01% (w/v) NaN3, pH 7.8. Store at 4°C.
- 4M KCl: Store at room temperature
- 1M MgCl2: Store at 4°C.
C. Conversion of Ca-Actin to Mg-Actin
- G-actin extraction. Add 270ml of cold X buffer
to 9 g acetone powder in a beaker on ice. Stir gently
with a glass rod to completely moisten the acetone
powder; excessive stirring will lead to increased α-
actinin extraction. Leave on ice for 30min, repeat stirring
every 10 min.
- Clarification. Centrifuge for 45min at 25,000g 4°C. Filter the supernatant through glass wool and
determine the volume after filtration.
- Polymerization/α-actinin elimination. Add
solid KCl to a final concentration of 3.3M, taking into
account a volume increase of about 10%. Stir at room
temperature until the solution has reached a temperature
of 15°C to allow complete solubilization of the
salt. Subsequently, chill the solution on ice without
stirring until its temperature has fallen back to 5°C. Centrifuge for 30min at 25,000g / 4°C. Discard the
F-actin/α-actinin pellet and filter the supernatant
through glass wool.
- Dialysis. Dialyse at 4°C overnight against 32
volumes of buffer Xb to bring [K+] to 0.1M.
- Tropomyosin elimination. Add 0.22 volumes of
4M KCl to bring [K+] to 0.8M. Stir for 90min at 4°C. Centrifuge for 3h 30min at 70,000g/4°C. Remove
tropomyosin-containing supernatant. Collect the
transparent F-actin pellets with a clean spatula and
pool into a small glass tissue grinder. To collect residual
actin, rinse centrifuge tubes with buffer X and
add to pool. Homogenize the F-actin solution on ice,
avoiding excessive foaming. Transfer to a 50-ml measuring
cylinder. Add 75 µl 1M MgCl2 and 375 µl
4M KCl and adjust volume to 38.6 ml with buffer X.
(If necessary, the protocol can be interrupted at this
point and the solution left to sit overnight at 4°C.) Repeat step 5 to remove tropomyosin completely, i.e.,
add 9ml 4M KCl, make up to 50ml with buffer X,
stir for 90min at 4°C, centrifuge for 3h 30min at
85,000g/4°C, and remove tromomyosin-containing
- Depolymerization. Resuspend and homogenize
the F-actin pellets as in step 5 in a total volume of
20-30ml buffer X. Dialyse overnight against 1 liter
- Sonication. Sonicate 2 × 10s in the dialysis bag.
Change dialysis buffer and continue dialysis for
- Clarification and gel filtration. Centrifuge the
actin for 2h at 250,000g / 4°C. Run the supernatant
over a Sephadex G-200 gel filtration column equilibrated
in buffer G at 15-20ml/h. Collect 5-ml fractions.
You should observe a first small and a second
large peak by following the A290; the second one is the
actin peak. Avoid collecting the peak front because of
possible CapZ contamination.
- Concentration determination. Pool actin fractions
and determine the concentration by spectrophotometry
(ε290nm = 26,600M-lcm-1) using the G buffer as
blank. Typically this will be 40-50 µM. Ca-G-actin can
be stored on ice for several weeks.
- 10 mM MgCl2
- 25 mM EGTA, pH 9.0
- Dilute Ca-G-actin to a concentration not higher than
10 µM with G buffer.
- Add MgCl2 to a final concentration of (x + 10) µM,
where x is the actin concentration in µM.
- Add EGTA to a final concentration of 200µM. Mix
gently by pipetting. The cation exchange will be
complete in about 3 min.
Actin can also be stored frozen or lyophilized, and
lyophilized actin is available commercially (Sigma A-
2522). However, these techniques lead to partial denaturation
of the protein. If actin is frozen or lyophilized,
sucrose should be added (2mg/ml per mg/ml actin),
and thawed or rehydrated actin should be dialysed
against buffer G and recycled by a cycle of polymerization,
high-speed sedimentation, and depolymerization.
The protein concentration should be
redetermined after this procedure.
Carlier, M.-E, Valentin-Ranc, C., Combeau, C., Fievez, S., and
Pantaloni, D. (1994). Actin polymerization: Regulation by divalent
metal ion and nucleotide binding, ATP hydrolysis and
binding of myosin. Adv. Exp. Med. Biol
Ebashi, S., and Maruyama, K. (1965). Preparation and some properties
of alpha-actinin-free actin. J. Biochem. (Tokyo) 58
Feuer, G., Molnar, E, Pettko, E., and Straub, E (1948). Studies on the
composition and polymerization of actin. Hung. Acta Physiol 1
Gershman, L., Newman, J., Selden, L., and Estes, J. (1984). Boundcation
exchange affects the lag phase in actin polymerization. Biochemistry 23
MacLean-Fletcher, S., and Pollard, T. (1980). Identification of a factor
in conventional muscle actin preparations which inhibits actin
filament self-association. Biophys. Res. Commun
Pardee, J., and Spudich, J. (1982). Purification of muscle actin. Methods Enzymol
Spudich, J., and Watt, S. (1971). The regulation of rabbit skeletal
muscle contraction. I. Biochemical studies of the interaction of
the tropomyosin-troponin complex with actin and the proteolytic
fragments of myosin. J. Biol. Chem