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        LABELING GC-GLOBULIN WITH FLUORESCEIN

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        1260

        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.

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