isolation of cell-surface and intracellular proteins from an astrocyte culture

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

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.

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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).
<ol>
	<li>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.</li>
	<li>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 &#62;70% confluent on the day the experiment is to take place. Ensure that cells are evenly distributed between dishes.</li>
	<li>At 16 h prior to assay, pipette laminin into culture medium to a final concentration of 24 nM, and incubate at 37 &#176;C.</li>
	<li>Immediately prior to assay, prepare the following, and then place on ice or refrigerate: CM-PBS (100 mg/L MgCl2&#8729;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&#160;[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).</li>
	<li>Remove dishes holding the astrocyte cultures from the incubator and discard the medium.</li>
	<li>Wash cells thrice with 4 mL chilled CM-PBS and place the dishes on crushed ice.</li>
	<li>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.</li>
	<li>Remove the biotin buffer using an aspirator and replace it with 4 mL quenching buffer. Leave on ice for 10 min.</li>
	<li>Aspirate the quenching buffer and replace it with an equivalent volume of the same. Again, leave on ice for 10 min.</li>
	<li>Discard the quenching buffer, and wash cells thrice with 4 mL chilled CM-PBS.</li>
	<li>Scrape cells into 1 mL chilled CM-PBS using a cell lifter and transfer the suspension to microcentrifuge tube.</li>
	<li>Pellet cells by centrifugation at 100 x g for 3 min. Discard the supernatant and re-suspend cells in 500 &#181;L of lysis buffer.</li>
	<li>Leave samples on ice for 30 min, vortexing every 5 min, or place them on an end-over-end rotator at 4 &#176;C.</li>
	<li>Centrifuge the lysate at 14,000 x g for 10 min at 4 &#176;C to pellet any detergent-insoluble materials. Transfer the supernatant into a new microcentrifuge tube.
	<ol>
		<li>Save 50 &#181;L of this lysate and add loading buffer to it. Then denature it by heating at 95 &#176;C in a dry bath; this is the &#34;input&#34; fraction, containing both biotinylated cell-surface proteins, as well as non-biotinylated cytosolic proteins.</li>
	</ol>
	</li>
	<li>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 &#181;L of streptavidin-agarose beads (normally stored at 4 &#176;C) to the lysate, and incubate at 4 &#176;C for 3 h on shaker/rocker.
	<ol>
		<li>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 &#181;L of the suspension into each sample.</li>
	</ol>
	</li>
	<li>Pellet streptavidin-agarose beads by centrifugation at 1500 x g for 30 s at 4 &#176;C.</li>
	<li>Save 50 &#181;L of the supernatant (add loading buffer and denature it at 95 &#176;C in a water bath or heating block); this represents the &#34;intracellular&#34; fraction and is comprised primarily of non-biotinylated cytosolic proteins.</li>
	<li>Resuspend the pelleted beads in 1 mL wash buffer, and rock this for 3 min at 4 &#176;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.</li>
	<li>Pellet the beads by centrifugation (1500 x g for 30 s at 4 &#176;C) and discard the overlying wash buffer. Add 50 &#181;L of 1X loading buffer (diluted using lysis buffer). Release biotin and streptavidin from beads by denaturing this at 95 &#176;C; this fraction should contain biotinylated cell-surface proteins only (&#34;cell-surface&#34; fraction).
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>

Source: Tham, D. K. L., et al. <a href="https://app.jove.com/v/55974">Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation.</a> J. Vis. Exp. (2017).
This video demonstrates the isolation of cell-surface and intracellular proteins from an astrocyte culture. The astrocyte culture is placed on ice to inhibit endocytosis, and then the cell-surface proteins are labeled with a biotinylation reagent. The cells are lysed to release the biotinylated cell-surface proteins and non-biotinylated intracellular proteins. Finally, streptavidin-coated beads are used to separate the biotinylated cell-surface proteins from the intracellular proteins.

1. Determining Relative Cell-surface Protein Expression in Astrocytes by Biotinylation
NOTE: Here, we illustrate the application of this biotinylation ...

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.

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

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

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