F-actin co-sedimentation assay
Goode Lab (3-26-04)
1. Thaw rabbit muscle actin (RMA) on ice overnight or rapidly in hand, then place on
ice. The latter method creates many short actin nuclei that greatly accelerate
subsequent polymerization. However, for co-sedimentation assays, this is not a
problem since one uses assembled actin.
2. To polymerize the actin, first add G-buffer to dilute actin to 20µM. Then add
initiation mix (IM) from the 20X stock to 1X final concentration (e.g. add 4µl 20xIM
to 76µl of actin). Mix well by pipeting up and down. Incubate at room temperature
(25C) for 1 hr. Actin is now polymerized and at steady state (i.e. distributed ~
0.2µM actin monomers and 19.8µM actin in polymer). Assembled actin is very
stable and can be stored at 4C and used over the course of several days, even longer
if one adds fresh ATP + DTT ) every few days (0.1mM of each).
3. Set up co-sedimentation reactions. MAKE SURE YOU ARE USING THE SAME
PIPETMAN FOR EVERYTHING. Since you do not know the relative affinity of
your protein for F-actin, initially try a range of concentrations of F-actin
(0,1,2,4,8,16µM) and a fixed concentration of your protein (e.g. 0.5µM).
Alternatively, use a set volume of in vitro translated [S35 methionine labeled] protein
that is easily detected by autoradiography (usually 2µl). Set up 20µl reactions in the
TLA 100 tubes (polycarbonate, 7 x 20mm tubes, Beckman #343775). Each reaction is
comprised of 18µl of F-actin and/or F-buffer (G-buffer + 1xIM) and 2µl of protein.
Be sure to include a "0" F-actin reaction to assess how much of your protein pellets
on its own. It is a good idea also to preclear your protein before adding to reactions
(use same centrifugation conditions below in step 4).
4. Pellet F-actin. Incubate reactions at room temperature for 10 minutes, then centrifuge
in a TLA100 rotor, 90000rpm, 20 min, 25C.
5. Sample preparation and analysis. Carefully remove supernatant (20µl) and mix with
10µl of 3x sample buffer in another tube. Boil the sample 2 min and let tube rest at
room temperature until you load gel. For radioactive samples, don't boil. Resuspend
pellet in 30µl 1X sample buffer, boil 2 min, and rest at room temperature. Load equal
volumes (e.g. 12 or 15µl) of pellets and supernatants on an SDS-PAGE gel (usually
10 or 12%). Coomassie stain gel to see actin and your protein. Subsequently, dry gel
and expose to film if monitoring co-sedimentation of an S35 met labeled protein.
6. Plot data as %protein bound vs. concentration of F-actin in reactions. The positive
control for the assay that we included (Crn1p, yeast coronin protein) should cosediment virtually 100% at concentrations equal to or above 1µM F-actin.