Actin Pelleting Assay
At relatively high centripetal forces, filamentous F-actin will pellet while globular G-actin will remain suspended in solution. This biophysical property can be leveraged in probing for actin-associated proteins and assessing actin polymerization competency. This protocol outlines how to fractionate actin protein solutions by centrifugation and probe the resulting pellet and supernatant fractions.
Follow Steps 1-6 of the Polymerizing Actin Protein protocol to generate F-actin filaments.
Spin the F-actin solution at 48,000 rpm (100,000 x g) for 1 hour at 26°C in an ultracentrifuge rotor (i.e. TLA-100).
3. Collect Fractions
Collect the supernatant fraction and add an equal volume of 2X Sample Buffer.
Gently wash the pellet twice with Actin Working Buffer.
Resuspend the pellet in 1X Sample Buffer to a final volume matching that of the supernatant fraction.
Analyze the pellet and supernatant fractions by SDS-PAGE. Polymerization reactions devoid of KCl and MgCl2 can serve as a negative control.