Assessing the Internalization of Target Surface Proteins Using a Biotin Derivative

Treat mouse cortical astrocytes with a cleavable biotin derivative.

Incubate at a low temperature to prevent protein internalization and facilitate biotin attachment to the target surface proteins.

Wash and add a warm medium, then incubate to induce internalization of the biotinylated proteins.

Wash and treat with a membrane-impermeable reducing agent to cleave the non-internalized biotin.

Add a quenching reagent to stop the reaction and wash.

Harvest the cells and centrifuge. Remove the supernatant.

Add a lysis buffer to lyse the cells, releasing the non-biotinylated and internalized biotinylated proteins.

Centrifuge to pellet the cell debris.

Collect the supernatant containing proteins, add streptavidin-agarose beads, and mix to capture the biotinylated proteins.

Centrifuge to pellet the beads and discard the supernatant.

Add a loading buffer and heat to denature and release the proteins from the beads.

Run the protein on a gel and transfer it onto a blotting membrane.

Detect using primary and secondary antibodies to visualize a distinct protein band, confirming successful protein internalization.

First, remove the astrocyte cultures from the incubator and aspirate the medium. Then, wash the cells thrice with 4 milliliters of chilled CM-PBS and place the dishes on crushed ice. Aspirate the CM-PBS and then pipette 3 milliliters of biotin buffer into each dish. Tilt the dishes back and forth a few times to ensure that buffer is well-distributed, and leave them on ice for 30 minutes.

Then, aspirate the biotin buffer and replace it with 5 milliliters of warm medium. Incubate one culture dish at 37 degrees Celsius for 15 minutes and a second dish at the same temperature for 30 minutes. Leave another dish at 4 degrees Celsius as the zero-minute sample.

At the end of the incubation period, discard the medium and wash the cells thrice with 4 milliliters of chilled CM-PBS. Afterwards, aspirate the CM-PBS and then pipette 6 milliliters of reducing buffer over the cells, and leave the sample on ice for 15 minutes.

Then, replace the medium with 6 milliliters of fresh reducing buffer and place the sample on ice for an additional 15 minutes.

This step is critical as the glutathione contained within the reducing buffer will cleave the biotin moieties remaining at the cell surface. This ensures that only internalized proteins will be biased and labeled.

Following that, remove the reducing solution and replace it with 6 milliliters of quenching buffer. Leave the sample on ice for another 15 minutes and repeat the quenching step once more. Then, discard the quenching buffer and wash the cells thrice with 4 milliliters of chilled PBS.

Aspirate the CM-PBS and then, scrape the cells into 1 milliliter of chilled PBS using a cell lifter, and transfer the suspension into a microcentrifuge tube. Pellet the cells by centrifugation at 100 g for three minutes. After three minutes, discard the supernatant and resuspend the cells in 500 microliters of lysis buffer.

Leave the sample on ice for 30 minutes and vortex every five minutes. Centrifuge the lysate at 14,000 g for 10 minutes at 4 degrees Celsius to pellet the detergent and soluble materials. Then, transfer the supernatant to a new microcentrifuge tube.

Using a cut pipette tip, add 150 microliters of the streptavidin agarose slurry to the lysate and incubate it at 4 degrees Celsius for three hours on a shaker. After three hours, pellet the streptavidin agarose beads by centrifugation at 1,500 g for 30 seconds at 4 degrees Celsius. Resuspend the beads in 1 milliliter of wash buffer and rock them 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. Then, pellet the beads by centrifugation at 1,500 g for 30 seconds at 4 degrees Celsius. Discard the overlying wash buffer, and add 50 microliters of 1x loading buffer.

Release biotin and streptavidin from the beads by denaturing at 95 degrees Celsius. This fraction should contain internalized cell surface proteins only. Then, separate the input, cell surface, and unbound fractions by SDS-PAGE and analyze by Western blotting.