Protocol

Adhesion Protein Protocols

Volume 1046 of the series Methods in Molecular Biology pp 273-293

Date:

Measurement and Analysis of In Vitro Actin Polymerization

  • Lynda K. DoolittleAffiliated withDepartment of Biophysics, UT Southwestern Medical Center and Howard Hughes Medical Institute
  • , Michael K. RosenAffiliated withDepartment of Biophysics, UT Southwestern Medical Center and Howard Hughes Medical Institute
  • , Shae B. PadrickAffiliated withDepartment of Biophysics, UT Southwestern Medical Center and Howard Hughes Medical Institute

* Final gross prices may vary according to local VAT.

Get Access

Abstract

The polymerization of actin underlies force generation in numerous cellular processes. While actin polymerization can occur spontaneously, cells maintain control over this important process by preventing actin filament nucleation and then allowing stimulated polymerization and elongation by several regulated factors. Actin polymerization, regulated nucleation, and controlled elongation activities can be reconstituted in vitro, and used to probe the signaling cascades cells use to control when and where actin polymerization occurs. Introducing a pyrene fluorophore allows detection of filament formation by an increase in pyrene fluorescence. This method has been used for many years and continues to be broadly used, owing to its simplicity and flexibility. Here we describe how to perform and analyze these in vitro actin polymerization assays, with an emphasis on extracting useful descriptive parameters from kinetic data.

Key words

Actin polymerization In vitro reconstitution Biochemical assay Quantitative analysis