Antibodies or other proteins must have a free thiol group in order to obtain immobilized enzymatic activity. This group can be introduced by specific reagents, and the reaction with S-acetylphobic succinic anhydride is described here. Source: Laboratory Manual of Enzymology

Operation method

basic program

Materials and Instruments

Antibody or protein solutions
AMSA solution Potassium phosphate Nitrogen Hydroxylamine EDTA
Cross-linked dextran G-25 columns

Move

See "Other" for "Reagents" required for the experiment.

Add 0.1 ml of AMSA solution per 1 ml of antibody or protein solution under nitrogen, seal the tube and stir slowly at room temperature for 0.5 h. To remove the acetyl group and expose the thiol groups, add 0.2 ml of 0.5 mol/L hydroxylamine dissolved in 0.1 mol/L pH 7.0 Tris-HCl, 0.01 mol/L EDTA and adjust to pH 7.0. Place at 30 °C for 4 min. Place at 30°C for 4 min. The mixture was passed through a column (cross-linked dextran) and eluted with 0.1 mol/L pH 6.0 potassium dihydrogen phosphate and 5 mmol/L EDTA. The protein fraction was collected (absorption measured at 280 nm) and concentrated to 1 ml by ultrafiltration.

Common Problems

Reagents:

AMSA solution (S-acetylphobic succinic anhydride; Mr = 174.2; 60 mg dissolved in 1 ml N,N'-dimethylformamide)

0.1 mol/L potassium dihydrogen phosphate, pH 7.0

Antibody or protein solution (IgG, 10 mg/ml dissolved in 0.1 mol/L potassium dihydrogen phosphate, pH 7.0)

Nitrogen

0.5 mol/L hydroxylamine ( _NH2OH-HCl, Mr = 69.5), 0.01 mol/L EDTA (Mr = 292.3) dissolved in 0.1 mol/L Tris-HCl pH 7.0 (3.46 g _NH2OH-HCl, 0.29 g EDTA dissolved in 100 ml 0.1 mol/L potassium dihydrogen phosphate pH 7.0)

Cross-linked dextran G-25 column, approximately 1.5 cm × 30 cm, equilibrated with 0.1 mol/L pH 6.0 potassium dihydrogen phosphate

5 mmol/L EDTA

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