Preparation Of Membranes By Dounce Homogenization

Purification of membranes prior to their solubilization may be desirable for some experiments. This procedure is a significant purification step in that it removes cytosolic proteins and nuclei prior to solubilization of membrane proteins. Removal of nuclei prior to the addition of harsh detergents may allow increased solubilization of cytoskeletal-as-sociated proteins without the problems inherent in the solubilization of nuclei and subsequent release of chromatin.

This protocol and the alternate protocol that follows can be used to isolate total cellular membranes. Both involve the use of hypotonic swelling of cells prior to their disruption.

The basic protocol employs a Dounce homogenizer, which has been useful for a number of different lymphocyte types. The exact conditions, including the number of strokes required for optimal cellular disruption without breakage of nuclei, must be determined for each cell type. This can generally be accomplished by monitoring the procedure with the use of a phase-contrast microscope. The alternate protocol utilizes nitrogen cavitation and requires both a cavitation device as well as a source of compressed nitrogen gas.

Materials

Cultured cells

Phosphate-buffered saline (PBS; appendix2a), ice-cold Dounce buffer with protease inhibitors (see recipe), ice-cold Tonicity restoration buffer with protease inhibitors (see recipe) 0.5 M EDTA, pH 7.6

Triton X-100 lysis buffer with protease inhibitors (see recipe)

Dounce homogenizers with type B tight-fitting pestle (Kontes), chilled to 4°C

Phase-contrast microscope

10-ml polycarbonate ultracentrifuge tubes

Beckman Ti-70.1 or equivalent horizontal or fixed-angle ultracentrifuge rotor Potter-Elvehjem grinder (optional; Kontes) Triton X-100 lysis buffer with protease inhibitors

NOTE: Carry out all steps in this protocol at 4°C or on ice.

1. Harvest cultured cells, wash in ice-cold PBS, and centrifuge as in steps 1 and 2 of the first basic protocol.

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