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Polymerizing Actin
Overview
Globular G-actin protein polymerizes into filamentous F-actin when supplemented with KCl and MgCl2. F-actin can be stored at 4°C for several weeks with the addition of an antibacterial agent such as 0.05% sodium azide. As F-actin is relatively stable in comparison to G-actin, actin is preferably stored in its polymerized form. Adenosine triphosphate (ATP) and dithiothreitol (DTT) must also be included and the pH range kept between 6.5 and 8.5.
Protocols
1. Resuspend
Reconstitute the lyophilized actin protein by resuspending in ice-cold ultra-pure water to 3 mg/ml (for Ultra-Pure Actin) or 9 mg/ml (for Pure Actin).
NOTE :
Note that 1 mg actin protein is supplied as 3 mg powder (extra mass attributed to trehalose, a lyoprotectant), and reconstitution/dilution should be based on the actin protein concentration.
2. Dilute
Dilute the resuspended actin protein to 0.4 mg/ml with Actin Working Buffer. Add ATP to 0.2 mM and DTT to 0.5 mM.
3. Incubate
Incubate the reconstituted actin protein on ice for 1 hour to depolymerize any actin oligomers.
4. Clarify
Clarify the reconstituted actin protein to remove any protein aggregates. Spin at 14,000 rpm (21,000 x g) for 15 minutes at 4°C in a microcentrifuge. Collect the supernatant on ice in a clean microtube.
NOTE :
If desired, a more rigorous clarifying spin can be conducted by spinning at 60,000 rpm (150,000 x g) for 2 hours at 4˚C in an ultracentrfuge rotor (i.e. TLA 100).
5. Assemble
Assemble the polymerization reaction by adding 1/10th volume Actin Polymer Buffer (10X) to the clarified actin protein on ice. Add ATP to 1 mM.
6. Polymerize
Incubate the polymerization reaction at room temperature for 1 hour.
The F-actin is now ready for experimental use and can be stored at 4°C for several weeks with the addition of 0.05% sodium azide.
