1. Preparation of actin
The source of actin used in this assay was non-muscle human platelets (β-actin) obtained from Cytoskeleton Inc. Stock aliquots at 10 mg/ml are kept in the -70ºC freezer. For the assay, the actin is diluted to 0.4 mg/ml in a buffer containing 5 mM Tris pH 8, 0.2 mM CaCl2, 0.2 mM ATP and 0.5 mM DTT, centrifuged at 20,000g for 10 mins at 4ºC. The supernatant, which is monomeric actin is ready to be polymerized. Actin polymerization is induced by the addition 50 mM KCl, 1 mM ATP and 2 mM MgCl2. The polymerization occurs at room temperature for 1 hour.
2. Preparation of protein
In this assay, we are using the C-terminus of talin, which has been purified as a GST-tagged protein. The protein to be used in the assay is subjected to a high-speed centrifugation to pre-clear of aggregates prior to incubation with F-actin.
The protein is spun at 100,000g in a Beckman ultracentrifuge for 20 mins at 22ºC.
3. Actin-protein Incubation
The amount of actin and protein in an assay will vary. In this example we are using actin in excess. Normally, a molar ratio is calculated, eg. a 4:1 molar ratio of actin to protein.
In the following assay, the final reaction volume is 150 µl. The buffer in which the protein and F-actin are incubated will depend on the protein to be tested and on the conditions required. In this example, we use a buffer containing 10 mM Tris pH 7.0, 1 mM ATP, 0.2 mM DTT, 1 mM EGTA, 0.1 mM CaCl2 and 2 mM MgCl2. The protein and F-actin are incubated in the buffer for 1 hour at room temperature.
4. Sedimentation of F-actin
Following the incubation, the samples are spun at 100,000g at 22ºC. We use a Beckman ultracentrifuge in the video. The rotor is carefully removed so as not to disturb the pellets.
5. Analysis of protein co-sedimentation with F-actin
The supernatants are carefully removed by pipetting into an eppendorf tube and 5 X Laemmli SDS-PAGE sample buffer is added. An appropriate volume of 1 X Laemmli SDS-PAGE sample buffer is added to the pellets remaining, pipetted up and down, and transferred to new eppendorf tubes. The relative amounts of protein in the pellets and supernatants are analyzed following their separation by SDS-PAGE and Coomassie Blue staining of the gel or Western blotting.
In order to determine the specificity of protein interaction with actin, actin concentration-dependent co-sedimentations can be performed. For this sort of experiment, a fixed amount of the protein and a series of increasing amounts of actin are incubated, and analysis is carried out as described above.