Sucrose Fractionation of Castor Bean

Materials

Castor beans       Gradient former Sucrose solutions in 0.01 M EDTA, pH 7.5       33%, 44%, 50%, 57%, 60% (w/v)        Grinding Medium             0.4 M sucrose             0.165 M Tris-HCl buffer, pH 7.5             0.01 M KCl             0.01 M MgCl             0.01 M EDTA adjusted to pH 7.5             0.01 M dithiothreitol       Chopping board and knife or razor blade       Mortar and pestle       Cheesecloth       Refrigerated centrifuge (ultracentrifuge preferred) with swinging bucket rotor       Spectrophotometer and tubes       Hemacytometer and phase contrast microscope

Procedure

1.Germinate castor beans by soaking overnight, followed by germination in moist vermiculite, or paper towels, at 30° C.

Castor beans are poisonous and may be fatal if taken internally. Wear gloves while handling.

2.After approximately 5 days, remove the embryos and cotyledons and discard. Wash the endosperm in distilled water and chill on ice.