PELLETING ACTIN

Materials:

• Ultra-Pure Actin (Cat. No. 160101) or Pure Actin (Cat. No. 190101)
• Actin Working Buffer (Cat. No. 000102; 5 mM Tris-HCl and 0.2 mM CaCl2, pH 8.0)
• Actin Polymer Buffer (10X; Cat. No. 000103; 500 mM KCl and 20 mM MgCl2)
• ATP
• DTT
• 2X Sample Buffer

Equipment:

•  Microcentrifuge at 4°C
•  Spectrophotometer
•  Ultracentrifuge and rotor (i.e. Beckman TL-100 Tabletop Ultracentrifuge and TLA-100 rotor)
•  SDS-Page system

Technical Notes:

• Avoid diluting actin beyond its critical concentration (dependent on ionic strength)
• Boiled factions containing Sample Buffer can be stored at -20°C for several weeks

Protocol:

1. Reconstitute actin to 0.4 mg/ml (see respective protocol for details)

 

2. Polymerize actin (see respective protocol for details)

3. Pellet

a. Spin at 48,000 rpm for 1 hour at 26°C in an ultracentrifuge

4. Collect supernatant and pellet fractions

a. Collect the supernatant and determine protein concentration by Bradford assay or A280 (extinction coefficient = 26,600 M-1cm-1)

b. Add an equal volume of 2X Sample Buffer to the supernatant fraction

c. Resuspend the pellet in 1X Sample Buffer to a final volume matching that of the supernatant fraction

d. Boil both fractions and analyze by SDS-PAGE

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Figure 1: Ultra-Pure Actin is >90% polymerization-competent. Ultra-Pure Actin was polymerized in the absence (-PB) or presence (+PB) of Actin Polymer Buffer (10X, Cat. No. 000103; 50 mM KCl and 2 mM MgCl2) followed by centrifugation at 48k rpm for 1 hour. Pellet (P) and supernatant (S) fractions were collected and subjected to SDS-PAGE and Coomassie G250-staining. >90% of Ultra-Pure Actin was incorporated into filaments as determined by measuring the residual protein concentration in the supernatant fraction.