assessing the internalization of target surface proteins using a biotin derivative

Assessing the Internalization of Target Surface Proteins Using a Biotin Derivative

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.

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Assessing the Internalization of  Target Surface Proteins Using a Biotin Derivative

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Determining Internalization Rate of Cell-surface Proteins in Astrocytes by Biotinylation
NOTE: In the following, we describe a typical pulse-chase biotinylation experiment used in this instance to track the endocytosis of AQP4 in astrocytes. Specialized materials required include sulfo-NHS-SS-biotin, streptavidin-agarose resin, reduced glutathione, and iodoacetamide (see Table of Materials).
<ol>
	<li>Prepare cultures of mouse cortical astrocytes in 60-mm dishes using the methods outlined in the previous section. Ensure that cells are approximately 70% confluent on the day of the assay and that each dish contains an equivalent number of cells.</li>
	<li>Immediately prior to assay, prepare the following, and place on ice or refrigerate: CM-PBS or Phosphate buffered saline (100 mg/L MgCl2&#8729;6H2O and 100 mg/L CaCl2 in 1X PBS, pH7.4), biotin buffer (0.5 mg/mL sulfo-NHS-SS-biotin in CM-PBS), reducing buffer (50 mM reduced glutathione, 75 mM NaCl and 75 mM NaOH), quenching buffer (50 mM iodoacetamide, 1% BSA, in CM-PBS), lysis buffer (25 mM Tris, pH 7.4, 25 mM glycine, 150 mM NaCl and 5 mM EDTA or Ethylenediaminetetraacetic acid, 1% triton X-100, 1X protease inhibitor cocktail), 3X loading buffer (150 mM Tris, pH 6.8, 6% SDS or Sodium dodecyl sulfate, 30% glycerol, 300 mM DTT or Dithiothreitol 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>Prepare fresh cell culture medium (Dulbecco&#39;s Modified Eagle Medium; or DMEM supplemented with 10% Fetal Bovine Serum (FBS), 1% penicillin/streptomycin, and 1% L-glutamine), and place it in a 37 &#176;C water bath.</li>
	<li>Remove astrocyte cultures from the incubator, and aspirate the medium using an aspirator.</li>
	<li>Wash cells thrice with 4 mL chilled CM-PBS, and place the dishes on crushed ice.</li>
	<li>Pipette 3 mL biotin buffer into each dish, tilt dishes back and forth a few times to ensure that the buffer is well-distributed, and leave on ice for 30 minutes.</li>
	<li>Aspirate biotin buffer, and replace it with 5 mL warm medium. Incubate a culture dish at 37 &#176;C for 15 min, and a second dish at the same temperature for 30 min. Leave another dish at 4 &#176;C as the 0 min sample.</li>
	<li>At the end of the incubation period, discard the medium and wash the cells thrice with 4 mL chilled CM-PBS. Pipette 6 mL reducing buffer over the cells and leave them on ice for 15 min.</li>
	<li>Remove the reducing buffer and replace it with 6 mL of fresh reducing buffer. Place the mixture on ice for an additional 15 minutes.</li>
	<li>Remove the reducing solution and replace it with 6 mL quenching buffer. Leave on ice for 15 min.</li>
	<li>Repeat the quenching step once more.</li>
	<li>Discard quenching buffer, and wash cells thrice with 4 mL chilled PBS.</li>
	<li>Using a cell lifter, scrape cells into 1 mL chilled PBS and transfer the suspension into a microcentrifuge tube.</li>
	<li>Pellet cells by centrifugation at 100 x g for 3 min. Discard the supernatant, and resuspend cells in 500 &#181;L lysis buffer.</li>
	<li>Leave this on ice for 30 min and vortex every 5 min. Alternatively, place the samples on an end-over-end rotator at 4 &#176;C for this duration.</li>
	<li>Centrifuge the lysate at 14,000 x g for 10 min at 4 &#176;C to pellet the detergent-insoluble materials, then transfer the supernatant to a new microcentrifuge tube. Save 50 &#181;L of this lysate, add loading buffer to it, and denature at 95 &#176;C in a dry bath; this is the &#34;input&#34; fraction, containing both biotinylated endocytosed proteins and nonbiotinylated proteins.</li>
	<li>Using a cut pipette tip, add 150 &#181;L of the streptavidin-agarose slurry to the lysate, and incubate at 4 &#176;C for 3 h on a shaker/rocker.</li>
	<li>Pellet streptavidin-agarose beads by centrifugation at 1,500 x g for 30 s at 4 &#176;C.</li>
	<li>Resuspend beads in 1 mL wash buffer and rock for 3 min at 4 &#176;C. Pellet beads (as per step 1.18) and discard the supernatant. Repeat this process 4 times to minimize the nonspecific binding of nonbiotinylated cytosolic proteins.</li>
	<li>Pellet beads by centrifugation at 1,500 x g for 30 s at 4 &#176;C and discard the overlying wash buffer. Add 50 &#181;L 1X loading buffer (diluted using lysis buffer). Release biotin and streptavidin from beads by denaturing at 95 &#176;C; this fraction should contain internalized cell-surface proteins only (&#34;endocytosed&#34; fraction).</li>
	<li>Separate input, cell-surface, and unbound fractions by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed 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>

assessing the internalization of  target surface proteins using a biotin derivative

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 a method to assess target protein internalization in the mouse cortical astrocytes using biotinylation, followed by cell lysis, streptavidin-based protein extraction, denaturation, and Western blot analysis to confirm successful internalization.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Determining Internalization ...

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.

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Assessing the Internalization of Target Surface Proteins Using a Biotin Derivative

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