Falling ball assay – Viscometry of F-actin solutions
(Ann Moon, modified by BG and JH)
Materials
 50µl or100µl capillaries
 appropriately sized ball bearings
 magnet
 clay (or play-doh)
Actin concentration:
Use actin at approximate concentration of 300µg/ml (7.5µM assuming 1µM actin = 40
µg/ml). (Equivalents = 30µg/100µl reaction = 15µl of 2mg/ml actin stock per 100µl
reaction). This will give you a viscosity such that, using an incline of 15°, the ball bearing
will take about 20 sec to fall through a 100µl capillary tube. Use actin at lower
concentration if using 50µl capillary tubes, or adjust to suit your needs. If you have not
used a certain batch of actin before, it is a good idea to do a trial run with actin added at a
range of concentrations to see what gives the most appropriate viscosity.
NOTE: Aliquoted actin stocks are stored in G-buffer at –80°C. However, some
polymerization of actin occurs during the freeze/thaw process/. Thawing the actin
overnight at 4°C or on ice helps to convert F-actin to G-actin.
Procedure:
Dilute actin in G-buffer to appropriate
concentration. Add 80µl of this actin
stock to each eppendorf tube. Add 10µl
of protein (or buffer alone) to each tube.
Add 10µl of 10X IM (initiation mix),
pipet up and down to mix, draw solution
up into capillary tube and plug bottom
with clay. I find it convenient to let
capillary tube rest in the eppendorf tube
that used to hold the solution. Continue until samples are done.
Reaction should polymerize in the tube for one hour. Then mount the tube on the inclined
plane using clay. Pick up ball bearing by pressing with finger and drop into the top of
capillary tube. Ball has a hard time falling through meniscus, so you need to draw it
through with a magnet, then hold the ball just under the surface. Start timing when you
release the ball and stop timing when the ball reaches the bottom.
If you accidentally let the ball fall down the tube before you are ready, this reaction
must be discarded – Do not attempt to get a reading by dragging the ball back to the
starting line (or flipping the tube around). When the ball rolls through the solution, it
breaks and removes a lot of the F-actin.