1. Determining Relative Cell-surface Protein Expression in Astrocytes by Biotinylation
NOTE: Here, we illustrate the application of this biotinylation technique to the study of the effects of the extracellular matrix molecule laminin on the cell-surface localization of the water-permeable channel aquaporin-4 (AQP4). Specialized materials required for this assay include sulfo-NHS-LC-biotin (sulfosuccinimidyl 6-(biotinamido) Hexanoate) and streptavidin-agarose resin (see Table of Materials).
- Using the method, prepare cultures of cortical astrocytes approximately 2 weeks in advance of the assay, and grow them in 75 cm2 vented culture flasks. When astrocytes are 80 - 90% confluent, detach them from the culture surface using 0.05% trypsin, and then passage them 1:3.
- At 48 h prior to the assay, when cells are again 80 - 90% confluent, passage astrocytes 1:3 (accounting for the change in the culture format) onto 60 mm cell culture dishes so that they are >70% confluent on the day the experiment is to take place. Ensure that cells are evenly distributed between dishes.
- At 16 h prior to assay, pipette laminin into culture medium to a final concentration of 24 nM, and incubate at 37 °C.
- Immediately prior to assay, prepare the following, and then place on ice or refrigerate: CM-PBS (100 mg/L MgCl2∙6H2O and 100 mg/L CaCl2 in 1X phosphate-buffered saline [PBS], pH 7.4), biotin buffer (0.5 mg/mL sulfo-NHS-LC-biotin in CM-PBS), quenching buffer (50 mM NH4Cl in CM-PBS), lysis buffer (25 mM Tris, pH 7.4, 25 mM glycine, 150 mM NaCl and 5 mM ethylenediaminetetraacetic acid [EDTA], 1% triton X-100, 1X protease inhibitor cocktail), 3X loading buffer (150 mM Tris, pH 6.8, 6% sodium dodecyl sulfate [SDS], 30% glycerol, 300 mM dithiothreitol [DTT] and 0.01% bromophenol blue), and wash buffer (10 mM Tris (pH 7.4), 1.5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1X protease inhibitor cocktail).
- Remove dishes holding the astrocyte cultures from the incubator and discard the medium.
- Wash cells thrice with 4 mL chilled CM-PBS and place the dishes on crushed ice.
- Pipette 2 mL biotin buffer into each well, and gently tilt dishes back and forth a few times to ensure complete coverage. Leave on ice for 30 min.
- Remove the biotin buffer using an aspirator and replace it with 4 mL quenching buffer. Leave on ice for 10 min.
- Aspirate the quenching buffer and replace it with an equivalent volume of the same. Again, leave on ice for 10 min.
- Discard the quenching buffer, and wash cells thrice with 4 mL chilled CM-PBS.
- Scrape cells into 1 mL chilled CM-PBS using a cell lifter and transfer the suspension to microcentrifuge tube.
- Pellet cells by centrifugation at 100 x g for 3 min. Discard the supernatant and re-suspend cells in 500 µL of lysis buffer.
- Leave samples on ice for 30 min, vortexing every 5 min, or place them on an end-over-end rotator at 4 °C.
- Centrifuge the lysate at 14,000 x g for 10 min at 4 °C to pellet any detergent-insoluble materials. Transfer the supernatant into a new microcentrifuge tube.
- Save 50 µL of this lysate and add loading buffer to it. Then denature it by heating at 95 °C in a dry bath; this is the "input" fraction, containing both biotinylated cell-surface proteins, as well as non-biotinylated cytosolic proteins.
- Widen the opening of a pipette tip by cutting off approximately 0.5 cm of material from its end using a pair of sharp scissors. Using this pipette tip, transfer 75 µL of streptavidin-agarose beads (normally stored at 4 °C) to the lysate, and incubate at 4 °C for 3 h on shaker/rocker.
- As streptavidin-agarose is frequently sold as a slurry containing 50% beads by volume, suspended in an antimicrobial solution, triturate the slurry to ensure that the beads are evenly suspended, and then pipette 150 µL of the suspension into each sample.
- Pellet streptavidin-agarose beads by centrifugation at 1500 x g for 30 s at 4 °C.
- Save 50 µL of the supernatant (add loading buffer and denature it at 95 °C in a water bath or heating block); this represents the "intracellular" fraction and is comprised primarily of non-biotinylated cytosolic proteins.
- Resuspend the pelleted beads in 1 mL wash buffer, and rock this for 3 min at 4 °C. Pellet beads (as in step 1.16) and discard the supernatant. Repeat this process 4x to minimize the nonspecific binding of nonbiotinylated cytosolic proteins.
- Pellet the beads by centrifugation (1500 x g for 30 s at 4 °C) and discard the overlying wash buffer. Add 50 µL of 1X loading buffer (diluted using lysis buffer). Release biotin and streptavidin from beads by denaturing this at 95 °C; this fraction should contain biotinylated cell-surface proteins only ("cell-surface" fraction).
- Separate input, cell-surface, and intracellular fractions by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and analyze by western blotting.
NOTE: While we used a 4 - 20% precast gradient gel in our experiments, a 12 - 14% separating gel with a 4% stacking layer (each containing 0.1% SDS) should suffice for the proteins of interest in this study. A molecular weight standard of the appropriate size range should also be used. Note that there can sometimes be an observable upshift in the apparent molecular masses of biotinylated proteins.