logo_tagline
Address_600_150

RECONSTITUTING 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)
  • ATP
  • DTT
Equipment:
  •  Microcentrifuge at 4°C
  •  Spectrophotometer
Technical Notes:
  • Actin is unstable in the absence of ATP, a divalent cation, and DTT
  • Reconstituted actin is stable at 4°C for several weeks
  • Add sodium azide to 0.05% to prolong storage at 4°C
Protocol:
1. Resuspend

a. Resuspend to 3 mg/ml (Ultra-Pure Actin) or 9 mg/ml (Pure-Actin) in ice-cold Milli-Q water

*buffer conditions upon reconstitution with water will consist of 2 mM Tris-HCl, 0.2 mM CaCl2, 0.2 mM ATP, and 1 mM DTT, pH 8.0

*1 mg actin = 3 mg powder

b. Dilute to 0.4 mg/ml with Actin Working Buffer

c. Add ATP to 0.2 mM and DTT to 0.5 mM

d. Incubate on ice for 1 hour

 

2. Clarify

a. Clarify at 14,000 rpm for 15 minutes at 4°C in a microcentrifuge

b. Collect the supernatant on ice

 

3. Store

a. Measure actin concentration by Bradford assay or A280 (extinction coefficient = 26,600 M-1cm-1)

b. Add sodium azide to 0.05% and store at 4°C

*product is stable under these storage conditions for several weeks