Actin Binding & Bundling Sedimentation Assays
Needed Items:

Beckman Airfuge & tubes, 30^o rotor

Proteins all dialyzed in the same buffers

Highly concentrated F-actin: >100 uM (>4 mg/ml)

I. Binding assays for tropomyosin, villin or caldesmon:

1. Dialyze proteins in buffers which approximate physiological conditions:

150 mM KCl

0.2 mM EGTA

0.2 mM EDTA

10 mM Imidazole pH 7.0

0.2 mM DTT

EDTA is included since tropomyosin stocks at high concentration form filament-like structures in the presence of Mg. However, assays are conducted with 2.5 mM free Mg by adding MgCl2 to the assays.

2. Set up a table with each assay component and the final concentrations of the assay like that shown below:

Final volume = 100 uL

	Final [Actin]	Final [TM]	Final [Villin]	Final Buffer	Volumes to Add in Microliters:	TM	Villin	F-Actin
1	5 uM	0.5 uM	0	61.7		5	0	33.3
2	5 uM	1.0 uM	0	56.7		10	0	33.3
3	5 uM	0.5 uM	1 uM	49.2		5	12.5	33.3
4	5 uM	1.0 uM	1 uM	44.2		10	12.5	33.3
5	5 uM	0.5 uM	4 uM	11.7		5	50	33.3
6	5 uM	1.0 uM	4 uM	6.7		10	50	33.3

Main Stock [Actin] = 125 uMDiluted [F-actin] = 15 uM
Stock [TM] = 10 uM
Stock [Villin] = 8 uM

3. .Just prior to the assay, remove aliquots of the soluble proteins of greater volume than you need for the assay. Warm to the assay temperature, and spin at 27 psi for 30 minutes in the Airfuge at room temp. Carefully remove the clarified supernatants and place in a separate tube; you don't want to pipet up any protein which might have pelleted, so leave 2 mm in the bottom. This can be mixed in with the stock. If you have a denaturation problem, the protein concentration can change drastically, although this is unusual. If there is pelleted protein, you should repeat a protein assay of your clarified sample.

4. The dialysis buffer does not include 2 mM MgCl2 or 0.2 mM ATP. Since these components will not alter the F-actin, and the same volume of diluted F-actin will be added to each of the samples, make the F-actin stock with 3 X the final concentration of Mg & ATP.

5. Just prior to the start of the assay, make a diluted stock solution of:

15 uM F-actin: 29.4 uL 125 uM Buffer:193 ul

6.2 mM MgCl2: 15.2 uL 0.1 M

0.6 mM ATP 7.35 uL 20 mM Total volume = 245 ul

To dilute highly concentrated F-actin, first mix buffer, Mg & ATP. Then, cut off a pipet tip with a razor blade so that the viscosity of the F-actin solution does not have as large an affect upon accurate volume measurement. Vortex the stock F-actin solution; once the rigid gel has been broken remove an aliquot, wipe off the tip with a Kimwipe and mix into the measured buffer, pipetting up & down several times. Mix further by pipetting up & down several times with a pipet set near the total volume of the sample. Do not introduce bubbles.

6. Assays should be started within 2 minutes of each other. To do this mix the samples in numbered Airfuge tubes in the following order:
Buffer + TM + Villin
Start the assays by adding F-actin to each tube and mixing 3 times with the pipetman.

7. Incubate at room temperature for specified time. Place a mark on each tube and place the tube in the rotor with the mark centered on the top, outside.

8. Spin @ 27 psi (150,000 x g) for 30 min. As you remove each tube from the rotor check to see if the mark is still on the top outside. This marks the position of the pellet.

9. Carefully remove 50 uL from the top of each supernatant and place in marked eppendorf tubes. Carefully wick away the remaining liquid from the side opposite the pellet with a twisted Kimwipe. With imagination you should be able to see the pellets.

10. Add 100 ul of ddH2O to each pellet. Add 33.3 uL of 4X sample buffer to each pellet tube. Add 16.7 uL of 4X sample buffer to each supernatant sample. To facilitate solubilizing the pellets, place in the -20 freezer for 1 hour to overnight. Mix by pipetting up & down where the pellet should be & then transfer to an eppendorf tube. Add 0.5 uL of 1% bromphenol blue to each of the samples.

11. Heat the samples for 5 min. in a boiling water bath. Vortex gently.

12. .Load equal volumes of pellets and supers onto a microgel. If quantitation is desired, load the end lanes twice as the bands of the end lane widen.

II.Bundling Assays:

1. An assay for bundling is virtually identical to the binding assay except that low speed sedimentation is used at the end. Typically, bundles sediment at 10,000g in 15 min., but little to no F-actin will sediment. It is important to note that this is not really a quantitative assay for bundling, as it requires that large bundles of actin filaments be formed, and smaller bundles are not sedimented. However, it is still a useful means for estimation of extensive bundling. It is not as sensitive as viscometric means for measuring crosslinking of filaments.

2. After incubation of F-actin, spin at 10,000 g for 15 min. If possible, use a rotor will holds the tubes horizontally so that pellets are in the very bottom of the tube. The tricky part of bundling assays is that the pellet is very soft, and easily disturbed. We use a Beckman microfuge for these assays.

3. Remove 50 uL of supernatant as before. Wick the remaining supernatant by inverting tube and letting the liquid run to the wick. Try & never touch the pellet. Resuspend pellets as described above & run gels.

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