Materials
1.200 mM K-borate,pH 9.0,10 ml.
2.100 mM lysine,100 mM K-borate,pH 9.0,1 ml aliquots stored at -20℃.
3.2 mM Tris-HCl,pH 8.5,4℃,250 ml.
4.100 mM DTT stock.
5.Centricon-30 concentrator.
6.5 mM Tris-acetate,0.1 mM DTT,pH 6.95,4℃,250 ml.
7.TRITC,10% on celite (Research Organics).
8.Bio-Beads SM-2 in 0.7x15 column.
Procedure (perform under reduced light)
1.Get 1-2 mg vinculin,stored in 2 mM Pipes,0.02% Azide,pH 7.6,in liquid nitrogen.Thaw quickly in warm water and chill in ice.
2.Mix vinculin solution 1:1 with 200 mM K-borate pH 9.0.Add 1.2 mg TRITC on celite per mg vinculin.
3.Stir on ice for 2 hr in the dark.
4.Pellet celite by centrifuging in a 42.2Ti rotor at 4℃,25,000 rpm for 10 min.
5.Collect supernatant and add equal volume of 100 mM lysine.Incubate for 2 hr on ice in the dark.
6.Equilibrate SM-2 column with buffer 3.
7.Apply the solution to the SM-2 column.Elute slowly.Pool fractions that appear pink in roomlight.
8.Measure volume,add DTT to 10 mM.
9.Concentrate to about 100μl (for 2 mg vinculin)using a Centricon-30 (concentrate in a SS34 rotor at 6,500 rpm,4℃ for about 20 min.Then collect at 2,000 rpm for 3 min).
10.Dialyze overnight against buffer 6 in the cold room.
11.Clarify at 25,000 rpm,4℃,for 20 min in a 42.2Ti rotor.
12.Determine the concentration of vinculin using Lowry assay.Determine dye/protein molar ratio by diluting conjugate 1:41 (10μl in 400μl of injection buffer)and reading OD555.
D/P = {OD555 x 41 / 60,000} / {(mg/ml)/ 130,000}
13.Dilute to 5 mg/ml for microinjection if necessary.Extra conjugated vinculin can be stored as aliquots in liquid N2.
