Mechanical Properties of Actin Filament Networks Depend on Preparation, Polymerization Conditions, and Storage of Actin Monomers

AbstractThis study investigates possible sources for the variance of more than two orders of magnitude in the published values for the shear moduli of purified actin filaments. Two types of forced oscillatory rheometers used in some of our previous work agree within a factor of three for identical samples. Polymers assembled in EGTA and Mg2+ from fresh, gel-filtered ATP-actin at 1mg/ml typically have an elastic storage modulus (G′) of ∼1Pa at a deformation frequency of 0.1–1Hz. G′ is slightly higher when actin is polymerized in KCl with Ca2+ and Mg2+. Gel filtration removes minor contaminants from actin but has little effect on G′ for most preparations of actin from acetone powder. Storage of actin monomers without frequent changes of buffer containing fresh ATP and dithiothreitol can result in changes that increase the G′ of filaments by more than a factor of 10. Frozen storage can preserve the properties of monomeric actin, but care is necessary to prevent protein denaturation or aggregation due to freezing or thawing