反应详情

The test compound swainsonine is prepared by 0.4 serial dilution of a 40 μM stock. Present in each determination is 10 μl diluted test compound, 25 μl of 10 mM paranitrophenyl mannopyranoside, 200 mM sodium acetate, pH 5.6 and 15 μl of purified rat liver Golgi mannosidase II. After incubating the reaction for 60 minutes at 37° C., the reaction is quenched with 50 μl of 0.5 M sodium carbonate. Absorption is read at 405 mn. After subtracting the blank from positive controls and samples, the samples are normalized against the positive control mean using a variable slope, sigmoidal curve fit, with bottom=0, top=100. The signal is proportional to the amount of products from the uninhibited reaction. The calculated IC50 for inhibition of purified Golgi mannosidase II by swainsonine hydrochloride is 0.068±0.021 μM.

1. customthe reaction for 60 minutes at 37° C.
2. customthe reaction is quenched with 50 μl of 0.5 M sodium carbonate
3. customAbsorption
4. customThe signal is proportional to the amount of products from the uninhibited reaction