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 and discard the medium by aspiration. Wash the cells thrice with 4 milliliters of chilled CM-PBS, and place the dishes on crushed ice.

Remove the CM-PBS by aspiration, and then, pipette two milliliters of biotin buffer into each well. Gently tilt the dishes back and forth a few times, to ensure complete coverage before placing the cultures on ice for 30 minutes.

Afterward, remove the biotin buffer, replace it with four milliliters of quenching buffer, and keep the cultures on ice for 10 minutes. Next, aspirate and replace with the same amount of the quenching buffer before leaving the cultures on ice for 10 minutes.

Then, discard the quenching buffer and wash the cells thrice with 4 milliliters of chilled CM-PBS. Following this, scrape the cells into one milliliter of chilled CM-PBS and transfer the suspension to a microcentrifuge tube.

Next, pellet the cells by centrifugation at 100 g for three minutes in a refrigerated microcentrifuge set to 4 degrees Celsius. After that, discard the supernatant, and resuspend the cells in 500 microliters of lysis buffer.

Then, leave the samples on ice for 30 minutes, vortexing every five minutes. Following that, centrifuge the lysate at 14,000 g for 10 minutes at 4 degrees Celsius to pellet any detergent and soluble materials. Then, transfer the supernatant into a new microcentrifuge tube. Save 50 microliters of the lysate and add 25 microliters of 3x loading buffer to it.

Subsequently, denature it by heating at 95 degrees Celsius in a dry bath. This is the input fraction containing both biotinylated cell surface proteins, as well as non-biotinylated cytosolic proteins. Using a cut pipette tip, transfer 75 microliters of streptavidin agarose beads to the lysate, and incubate it at 4 degrees Celsius for three hours on a shaker.

After that, pellet the streptavidin agarose beads by centrifugation at 1,500 g for 30 seconds at four degrees Celsius. Save 50 microliters of the supernatant, and add 25 microliters of 3x loading buffer. Then, denature it at 95 degrees Celsius in a water bath or a heating block. This represents the intracellular fraction and is comprised primarily of non-biotinylated cytosolic proteins.

Afterward, resuspend the pelleted beads in one milliliter of wash buffer, and rock it for three minutes at 4 degrees Celsius. Pellet the beads, and discard the supernatant. Repeat this process four more times to minimize the nonspecific binding of non-biotinylated cytosolic proteins.

Add 50 microliters of loading buffer diluted to 1x using lysis buffer. Release biotin and streptavidin from the beads by denaturing them at 95 degrees Celsius. This fraction should contain biotinylated cell surface proteins only.