LABELING GC-GLOBULIN WITH FLUORESCEIN

Materials

1.2 mM PIPES,pH 7.0,500 ml.

2.Gc-globulin (Calbiochem 345802),1 mg.

3.CFSE (Carboxyfluorescein succinimidyl ester; Molecular Probes).

4.Lysine,100 mM in buffer 1,10 ml.

5.G25-150 desalting column,1 cm diameter.Equilibrated with buffer 1.

6.Centricon-30.

7.PBS solution A,200 ml.

Procedure

1.Dissolve 1 mg Gc-globulin in 200μl buffer 1,dialyze overnight at 4℃.

2.Transfer Gc-globulin into a small vial with a flea bar.

3.Dissolve ~1 mg CFSE in DMSO at a concentration of 50 mg/ml.Dilute 1:10 into buffer 1.

4.Mix 50μl CFSE with the Gc-globulin solution while stirring.Let react at room temperature for 150 min in the dark (without stirring).

5.Stop reaction by addition lysine to 10 mM.

6.Desalt in a G25 column,collect fluorescent fractions in the void volume.

7.Concentrate with Centricon-30.

8.Clarify in a 42.2Ti rotor at 25,000 rpm,4oC for 30 min.Measure concentration with Lowry assay.

9.Dialyze into PBS solution A,can be stored at 4℃ for several weeks with 0.01% NaN₃,or frozen in liquid N₂.