The overall goal of this technique is to test the ability of a protein to co-sediment with filamentous actin, and if binding is observed, to measure the affinity of the interaction. This method is a straightforward technique to determine if a protein binds F-actin. The advantages of this technique are that it does not require specialized equipment aside from a ultracentrifuge, and all reagents can be made or purchased.
To begin, prepare high purity protein as described in the text protocol. Determine the protein concentration by measuring the absorbents at 280 nanometers. Just before use, hard spin the protein to remove aggregates of insoluble protein.
If solubility is a concern, remeasure the protein concentration after centrifugation. Next, remove an aliquot of globular actin or g-actin from the minus 80 degree celsius freezer and thaw it quickly. Add 10X polymerization buffer to the G-actin and dilute to a final concentration of 1X with water.
Incubate the mixture for one hour at room temperature to allow the actin to polymerize. After polymerization, store the polymerized filamentous actin, or F-actin, in solution at four degrees celsius, where it will be stable for a few weeks. Before using the F-actin after storage, gently invert or flick the tube several times to ensure that all actin is dissolved and uniformly distributed in solution.
Establish binding of the protein of interest to F-actin as described in the text protocol. Once F-actin binding has been established, serially dilute the protein to make a concentration series containing seven to eight samples at two times the final concentration to be tested. For example, if the range to test is 0.1 to eight micromolar, prepare dilutions of 16, eight, four, two, one, 0.5, and 0.2 micromolar protein in 1X reaction buffer in microfuge tubes.
Dilute the protein of interest to the final concentration in 1X reaction buffer in ultracentrifuge tubes. Add F-actin to the appropriate samples. Then incubate all of the samples for 30 minutes at room temperature.
After 30 minutes, remove one fifth of each sample and mix with sample buffer. These are the total samples. Load the ultracentrifuge sample tubes into the rotor and centrifuge for 20 minutes at 100, 000 times G and four degrees celsius.
After centrifugation, remove three quarters of the supernatant from each tube and mix with 4X sample buffer in a separate microfuge tube. These are the supernatant samples. Remove the remaining supernatant with a gel loading tip, taking care not to disturb the pellet.
Then add 1X sample buffer to all tubes and incubate for at least five minutes at room temperature. Following incubation triturate the sample eight to ten times with a p200 pipette tip, continually washing the pellet area of the tube. Gently scrape the pipette tip over the pellet during the trituration to help with re-suspension.
Transfer the re-suspended protein to a microfuge tube. These are the pellet samples. Finally, analyze the total and pellet samples by SDS page.
Image the coomassie stained gels using an imaging system that measures protein band intensities over a wide and linear range. Increasing concentrations of alpha e catenin homodimer were incubated in the presence or absence of 0.2 micromolar F-actin. The samples were centrifuged and the resulting pellets were analyzed.
As expected, the alpha e catenin homodimer co-sedimented with F-actin above background. BSA was run as a negative control. Bound protein was quantified and plotted over free protein to calculate the affinity of the interaction.
This technique can be done in four hours or less if F-actin is already polymerized. While attempting this procedure it is critical that the supernatant be removed quickly after centrifugation and the pellet re-suspended completely. As in all binding experiments it is critical that the binding to F-actin is saturated and that the purity plus F-actin plateaus, without a plateau it is not possible to calculate an accurate association equilibrium constant.
There are fundamental limitations to the co-sedimentation assay that researchers should be made aware of, most importantly, it does not produce a true equilibrium constant, and as a result can miscalculate or fail to detect low affinity interactions. Despite these limitations, the co-sedimentation assay is an accessible and effective tool to determine if a protein binds F-actin and to measure the affinity of the interaction.
