Actin Pelleting Assay

Actin pelleting assay graphical abstract

Overview

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.

Protocols

Polymerize F-actin filaments1. Polymerize

Follow Steps 1-6 of the Polymerizing Actin Protein protocol to generate F-actin filaments.

Rotate at 40,000 rpm 60 minutes2. Pellet

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).

Collect Fractions3. 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.

Unpolymerized and polymerized Reactions4. Analyze

Analyze the pellet and supernatant fractions by SDS-PAGE. Polymerization reactions devoid of KCl and MgCl2 can serve as a negative control.

Scroll to Top