Isolation of Cell-Surface and Intracellular Proteins from an Astrocyte Culture

Take an astrocyte culture and remove the medium. Add a physiological buffer and incubate on ice, inhibiting endocytosis of cell-surface proteins.

Incubate with a biotinylation reagent that binds the cell-surface proteins.

Next, incubate with a quenching buffer to inactivate any unreacted reagent.

Add a cold physiological buffer and mechanically detach the cells. Transfer the cells, then centrifuge and discard the supernatant.

Incubate with a lysis buffer to lyse the cells, releasing biotinylated cell-surface and non-biotinylated intracellular proteins.

Centrifuge, collect the supernatant, and separate a fraction with total protein content.

Incubate the remaining fraction under agitation with streptavidin-coated beads that bind biotinylated proteins.

Spin down the cell-surface protein-bound beads and separate the supernatant containing intracellular proteins.

Wash the beads under agitation to remove non-specifically bound proteins, then centrifuge and discard the supernatant, isolating the cell-surface proteins.

The fractions containing total, intracellular, and cell-surface proteins are ready for analysis.

To begin this procedure, remove the astrocyte cultures from the incubator an