Polymerizing and labeling actin

Department of Physics, Willamette University

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Polymerizing and labeling actin

1) Thaw out 10 uL aliquot of the 2 mg/mL G-actin stock (Cytoskeleton, Inc).

• MW actin: ~43 kDa

2) Add 30 uL of G-buffer to dilute sample to 0.5 mg/mL actin (a total volume of 40 uL)

3) Let sit on ice for at least 30 minutes

4) Add 3.5 uL of polymerization salts to the 40 uL sample

• Final concentration of salts in the actin: 1 mM ATP, 50 mM KCl, 2 mM MgCl2

5) Add ~0.5 uL of 300 uM stock of TRITC-phalloidin (from the -20 freezer)

RECIPES

• G-buffer: 2 mM Tris pH 8, 0.2 mM CaCl2, 1 mM DTT, 0.2 mM ATP

	1 mL G-buffer
1 M Tris, pH 8	2 uL
10 mM CaCl2	20 uL
100 mM ATP	2 uL
1 M DTT	1 uL
deionized water	975 uL

• Polymerization salts

	15.8 uL p-salts
100 mM ATP	2 uL
3 M KCl	3.3 uL
100 mM MgCl2	4 uL
deionized water	6.5 uL

• 10 mM CaCl2

	1 mL 10 mM CaCl2
1 M CaCl2	10 uL
deionized water	990 uL

• 100 mM MgCl2

	100 uL 100 mM MgCl2
1 M MgCl2	10 uL
deionized water	90 uL