Glycosidase Digestions

EndoH

Buffer (2x):

200 ml 750 mM NaCi, pH 5.5

200 ml 1 % Triton-X-100

8 ml 100 mM PMSF

7 ml 2-mercaptoethanol

585 ml water milliQ

Units required: 2 mU for 20 ml of B18 supernatant at overnight incubation

Protocol: Add 5 ml of 0.08 % SDS to up to 20 ml protein solution. Incubate for 2 min at 95°C. cool down on ice and add 20 ml of buffer plus 2 ml of endoH (= 2 mU). Incubate at 37°C for at least 6 h or better overnight. Optionally, you can omit the detergents (triton, SDS) and the reaction works nearly equally well.

EndoF

Buffer (2x): 993 ml 100 mM NaAc, 1 % NP40, pH 5.2

7 ml 2-mercaptoethanol

Units required: 40 munits for 20 ml of B18 supernatant at overnight incubation

Protocol: Add detergent and heat as above. Add buffer and enzyme as above.

PNGaseF

Buffer (2x): 993 ml 200 mM Na-phosphate, 20 mM EDTA, 1 % Triton-X-100

7 ml 2-mercaptoethanol

Units required: 200 munits for 20 ml of B18 supernatant at overnight incubation

Protocol: As above.

a-mannosidase

Buffer: 50 mM NaAc, 1 mM ZnCl2, pH 4.6

Units required: probably about 1 unit for 20 ml of B18 supernatant at overnight incubation. The enzyme has to be dialysed against the buffer

Protocol: Add 20 ml of 2 % SDS to 20 ml of protein solution. Incubate for 5 min at 95°C and cool down on ice for 5 min afterwards. Spin for 15 min at maximum speed and transfer the supernatant into a fresh Eppendorf tube. Dilute with the maximum amount of 160 ml of buffer and add the dialysed enzyme to give a total volume of 200 ml. Cover the reaction mixture with 200 ml of paraffin and incubate overnight. On the next day, acetone-precipitate.