This project was supported by the Canadian Institute of Health Research PG#20R47867.

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 and streptavidin-agarose resin (see Table of Materials).
<ol>
	<li>Using the method outlined in Noel et al.12, 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 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 EDTA, 1% triton X-100, 1X protease inhibitor cocktail), 3X loading buffer (150 mM Tris, pH 6.8, 6% SDS, 30% glycerol, 300 mM 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 with 4 mL quenching buffer. Leave on ice for 10 min.</li>
	<li>Aspirate the quenching buffer, and replace 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.
	<ol>
		<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>
	</ol>
	</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 13, and analyze by western blotting 14. 
		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>
2. Determining Internalization Rate of Cell-surface Proteins in Astrocytes by Biotinylation
NOTE: In the following, we describe a&#160;typical pulse-chase biotinylation experiment&#160;used in this instance to track the endocytosis of AQP4 in astrocytes. This method is based on that used by Madrid et al.15. Specialized materials required include sulfo-NHS-SS-biotin, streptavidin-agarose resin, reduced glutathione, and iodoacetamide (see the 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 (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, 1% triton X-100, 1X protease inhibitor cocktail), 3X loading buffer (150 mM Tris, pH 6.8, 6% SDS, 30% glycerol, 300 mM 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>Prepare fresh cell culture medium (DMEM supplemented with 10% Fetal Bbovine 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 buffer is well-distributed, and leave this on ice for 30 min.</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 medium, and wash cells thrice with 4 mL chilled CM-PBS. Pipette 6 mL reducing buffer over cells, and leave on ice for 15 min.</li>
	<li>Remove reducing buffer, and then replace with 6 mL fresh reducing buffer.&#160;Place on ice for an additional 15 min.</li>
	<li>Remove reducing solution and replace with 6 mL quenching buffer. Leave on ice for 15 min.</li>
	<li>Repeat quenching step once more.</li>
	<li>Discard quenching buffer, and wash cells thrice with 4 mL chilled PBS.</li>
	<li>Scrape cells into 1 mL chilled PBS using a cell lifter, 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 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, as well as 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 shaker/rocker. See 1.15 for additional details on this step.</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 2.18), and discard the supernatant. Repeat this process 4x 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 overlying wash buffer. Add 50 &#181;L 1X loading buffer (diluted&#160;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 SDS-PAGE13, and analyze by western blotting14. 
	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>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transferrin+uptake+and+release+by+reticulocytes+treated+with+proteolytic+enzymes+and+neuraminidase.">Transferrin uptake and release by reticulocytes treated with proteolytic enzymes and neuraminidase.</a> Biochim Biophys Acta. 426 (3), 385-398 (1976).</li><li>Katsura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+and+regulated+membrane+expression+of+aquaporin+1+and+aquaporin+2+water+channels+in+stably+transfected+LLC-PK1+epithelial+cells.">Constitutive and regulated membrane expression of aquaporin 1 and aquaporin 2 water channels in stably transfected LLC-PK1 epithelial cells.</a> Proc Natl Acad Sci U S A. 92 (16), 7212-7216 (1995).</li><li>Li, Y., Cam, J., Bu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-density+lipoprotein+receptor+family:+endocytosis+and+signal+transduction.">Low-density lipoprotein receptor family: endocytosis and signal transduction.</a> Mol Neurobiol. 23 (1), 53-67 (2001).</li><li>Gyorgy, P., Melville, D. B., Burk, D., Du Vigneaud, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+possible+identity+of+vitamin+H+with+biotin+and+coenzyme+R.">The possible identity of vitamin H with biotin and coenzyme R.</a> Science. 91 (2358), 243-245 (1940).</li><li>Cole, S. R., Ashman, L. K., Ey, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotinylation:+an+alternative+to+radioiodination+for+the+identification+of+cell+surface+antigens+in+immunoprecipitates.">Biotinylation: an alternative to radioiodination for the identification of cell surface antigens in immunoprecipitates.</a> Mol Immunol. 24 (7), 699-705 (1987).</li><li>Green, N. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proc Natl Acad Sci U S A. 76 (9), 4350-4354 (1979).</li><li>Madrid, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarized+trafficking+and+surface+expression+of+the+AQP4+water+channel+are+coordinated+by+serial+and+regulated+interactions+with+different+clathrin-adaptor+complexes.">Polarized trafficking and surface expression of the AQP4 water channel are coordinated by serial and regulated interactions with different clathrin-adaptor complexes.</a> EMBO J. 20 (24), 7008-7021 (2001).</li><li>Tham, D. 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The authors declare no conflict of interest.

Modifications: 
As these methods were designed for use with adherent cells, we have specified the use of PBS containing 100 mg/L MgCl2&#8729;6H2O and
100 mg/L CaCl2 (CM-PBS) for the washing steps and as the base of certain buffers so as to ensure that the cells remain attached to the culture surface and that cell-cell junctions are not disrupted. However, the protocols may also be applied to nonadherent cell types if the cells are pelleted...

Proteins at the cell surface play a variety of roles central to maintaining cell function. In numerous instances, their activity is dependent on, or modulated by endocytic processes that either temporarily sequester them in intracellular sites, or that direct them towards degradative pathways1,2,3,4,5. Here, we highlight 2 biotinylation-based approaches designed to allow the user to specifically tag and isolate proteins expressed at the plasma membrane, and those newly internalized. Via these methods, the cell-surface expression and endocytic rate of any protein of interest may be quantified, thus allowing a clearer assessment of its regulation to be achieved.
Determining relative cell-surface protein expression by biotinylation
Biotin, or vitamin B7, formerly known as vitamin H6, is a small water soluble molecule that can be used to chemically modify reactive amine, sulfhydryl, and carboxyl groups of biological molecules. The current crop of cell-surface biotinylation reagents consist primarily of membrane-impermeant sulfonated N-hydroxysuccinimide (sulfo-NHS) esters of biotin or its derivatives, designed to react with the amines present on the side-chains of lysine residues of proteins expressed at the cell surface when these become deprotonated under basic conditions, resulting in the latter forming an amide bond with the biotin moiety7. Thusly modified, cell-surface proteins may then be isolated via the use of avidin, a 66 - 69 kDa tetrameric protein possessing great affinity for biotin, binding to the latter with a dissociation constant of approximately 10-15, marking it as one of the strongest noncovalent interactions known8,9.
A number of alternative methods of quantifying protein expression at the cell surface have been used in previous studies. The labeling of unpermeabilized cells using fluorescently tagged antibodies specific for the protein of interest, followed by visualization via fluorescence microscopy, for instance, is a commonly employed approach, but is heavily reliant on the availability of antibodies that can bind to extracellular epitopes. More recently, methods involving the use of chimeric proteins bearing pH-sensitive fluorophores that react to being exposed to acidic media have also been successfully employed10. However, such assays usually involve the exogenous expression of these constructs in cell lines in which the protein of interest is not natively found. These approaches are nonetheless able to provide valuable information regarding the subcellular localization and exocytic itinerary of the target protein, and should therefore be used in conjunction with the biotinylation-based approaches described here if the tools are available.
In a typical biotinylation assay, the cells are first washed thoroughly in 4 &#176;C PBS. This removes any traces of serum proteins introduced by the culture medium, thus ensuring that these will not consume excess amounts of biotin in the next step. More importantly, the reduction in temperature causes endocytosis to decelerate significantly. The biotinylation reagent is then added. Next, the cells are washed again, and then incubated with a quenching buffer containing either glycine or NH4Cl, the purpose of which is to inactivate all remaining traces of unreacted biotin. The cells are then lysed, following which agarose-immobilized streptavidin is added to precipitate the biotinylated proteins. Analysis is commonly performed via western blotting, allowing the relative cell-surface expression of various proteins to be quantified.
Due to the basis of this assay, it is suitable for use only with proteins possessing portions exposed to the extracellular environment. Multipass transmembrane proteins, which likely possess a number of reactive lysines within their loop regions, are the most amenable to this method, while single-pass proteins tend to be less susceptible to being biotinylated. Even in these cases, there remains a possibility that conformational changes or intermolecular interactions may occlude certain reactive sites, resulting in a lower-than-expected biotinylation yield.
Determining internalization rate of cell-surface proteins by biotinylation
The principles of this assay are largely similar to those of cell-surface biotinylation, with a number of exceptions, the most important of which is the use of reversible biotinylation reagents. The biotin groups (of these) possess disulfide bonds, within their structures, that are susceptible to reducing agents; this is exploited to ensure that only cell-surface proteins taken into intracellular sites during the assay period will be left biotinylated. An assay generally takes place in the following manner. The cells are first washed and biotinylated with cold reagents, then cell culture medium at 37 &#176;C is re-introduced, and the cells are returned to the incubator; this causes the labeled cell-surface proteins to undergo endocytosis. The reducing agent glutathione - which cannot penetrate the membrane - is then added to break the disulfide bonds of the biotin moieties attached to proteins remaining on the cell surface. Finally, the broken disulfide bonds are reacted with iodoacetamide, consuming the labile thiol groups and preventing the bonds from reforming. As before, the cells are then lysed, and the labeled proteins are precipitated using streptavidin-agarose.
The limitations discussed in the previous section also apply here due to the similarities shared between the methods. In addition, it is worth bearing in mind that the temperature shifts involved in this assay preclude the exact determination of how much protein is endocytosed for each increment of time, particularly in the case of rapidly internalized or rapidly recycling proteins. The assay therefore only provides a semiquantitative estimation of endocytic rates. Total internal reflection fluorescence microscopy can be used to track the uptake of each loaded vesicle and provide a more precise measurement of the kinetics of endocytosis. It can therefore provide a very useful complement to this assay, assuming that a fluorescently tagged chimeric construct of the protein of interest is available11.

<table><tbody><tr><td>Ammonium chloride (NH&lt;sub&gt;4&lt;/sub&gt;Cl)</td><td>Fisher Scientific</td><td>A661-500</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A9647-50</td><td></td></tr><tr><td>Bromophenol blue</td><td>Bio-Rad</td><td>#1610404</td><td></td></tr><tr><td>cOmplete protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>11697498001</td><td></td></tr><tr><td>Disodium Ethylenediaminetetraacetate dihydrate (EDTA)</td><td>Bio-Rad</td><td>#1610729</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Bio-Rad</td><td>#1610611</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco/Thermo Fisher Scientific</td><td>11960-044</td><td></td></tr><tr><td>EZ-Link Sulfo-NHS-LC-Biotin</td><td>Thermo Fisher Scientific</td><td>#21335</td><td></td></tr><tr><td>EZ-Link Sulfo-NHS-SS-Biotin</td><td>Thermo Fisher Scientific</td><td>#21331</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco/Thermo Fisher Scientific</td><td>16000-044</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229-1</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G8898&amp;nbsp;</td><td></td></tr><tr><td>Iodoacetamide</td><td>Bio-Rad</td><td>#163-2109</td><td></td></tr><tr><td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td><td>Sigma-Aldrich</td><td>L2020</td><td>Thaw on ice.</td></tr><tr><td>L-glutamine</td><td>Gibco/Thermo Fisher Scientific</td><td>25030-081</td><td></td></tr><tr><td>Mouse monoclonal anti-&amp;beta;-actin antibody (AC-15)</td><td>Sigma-Aldrich</td><td>A5441</td><td></td></tr><tr><td>Mouse monoclonal anti-&amp;beta;-dystroglycan antibody (43DAG1/8D5)</td><td>Leica Biosystems</td><td>B-DG-CE</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Gibco/Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Peroxidase AffiniPure Donkey anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch Laboratories</td><td>715-035-150</td><td></td></tr><tr><td>Peroxidase AffiniPure Goat anti-Rabbit IgG (H+L)</td><td>Jackson ImmunoResearch Laboratories</td><td>111-035-045</td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Gibco/Thermo Fisher Scientific</td><td>10010-023</td><td></td></tr><tr><td>Reduced glutathione</td><td>Sigma-Aldrich</td><td>G6529</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Fisher Scientific</td><td>S271-500</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Sigma-Aldrich</td><td>862010&amp;nbsp;</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Fisher Scientific</td><td>S318-100</td><td></td></tr><tr><td>Streptavidin agarose resin</td><td>Thermo Fisher Scientific</td><td>#20347</td><td></td></tr><tr><td>Rabbit polyclonal anti-AQP4 antibody</td><td>Alomone</td><td>AQP-004</td><td></td></tr><tr><td>Tris base (Trizma base)</td><td>Fisher Scientific</td><td>BP152-1</td><td></td></tr><tr><td>Tris-HCl</td><td>Fisher Scientific</td><td>BP153-1</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>BP151-500</td><td></td></tr></tbody></table>

determining cell-surface expression and endocytic rate of proteins in primary astrocyte cultures using biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Using cell-surface biotinylation to assess the plasma membrane expression of AQP4 in astrocytes 
Laminin-treated astrocyte cultures and untreated control cells were subject to cell-surface biotinylation using the methods described. Biotinylated proteins were precipitated with agarose-conjugated streptavidin, and then separated via SDS-PAGE. Cell-surface fractions were probed for AQP4&#160;and &#946;-dystroglycan (&#946;-DG) as a cell-surface loading control, while th...

Watch this Scientific Journal Video about Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation at JoVE.com

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

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9.4K Views.  University of British Columbia. Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

Video: Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biology

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population of surface-expressed receptors is constantly adapting and subject to strict mechanisms of regulation. The paradigmatic example and one of the most studied trafficking events in biology is the regulated control of the synaptic expression of glutamate receptors (GluRs). GluRs mediate the vast majority of excitatory neurotransmission in the central nervous system and control physiological activity-dependent functional and structural changes at the synaptic and neuronal levels (e.g., synaptic plasticity). Modifications in the number, location, and subunit composition of surface expressed GluRs deeply affect neuronal function and, in fact, alterations in these factors are associated with different neuropathies. Presented here is a method to study GluR trafficking in dissociated hippocampal primary neurons. An &#34;antibody-feeding&#34; approach is used to differentially visualize GluR populations expressed at the surface and internal membranes. By labeling surface receptors on live cells and fixing them at different times to allow for receptors endocytosis and/or recycling, these trafficking processes can be evaluated and selectively studied. This is a versatile protocol that can be used in combination with pharmacological approaches or overexpression of altered receptors to gain valuable information about stimuli and molecular mechanisms affecting GluR trafficking. Similarly, it can be easily adapted to study other receptors or surface expressed proteins.

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Cell-surface Biotinylation Assay

A cell can regulate the amount of particular proteins on its cell membrane through endocytosis, following which cell surface proteins are effectively sequestered in the cytoplasm. Once within a cell, these surface proteins can be either destroyed or &#8220;recycled&#8221; back to the membrane. The cell surface biotinylation assay provides researchers with a way to study these phenomena. The technique makes use of a derivative of the small molecule biotin, which can label surface proteins and then be chemically cleaved. However, if the surface protein is endocytosed, the biotin derivative will be protected from cleavage. Thus, by analyzing the uncleaved, endocytosed biotin label, scientists can assess the amounts of internalized surface proteins.In this video, we review the concepts behind the biotinylation assay, delving into the chemical structure of the biotin derivative and the mechanism of its cleavage. This is followed by a generalized protocol of the technique, and finally, a description of how researchers are currently using it to study the dynamics of different cell surface proteins.

A cell can regulate the amount of particular proteins on its cell membrane through endocytosis, following which cell surface proteins are effectively ...

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

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.

Izolacja białek powierzchniowych i wewnątrzkomórkowych z hodowli astrocytów

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

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

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A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Cell-surface Biotinylation Assay

Izolacja białek powierzchniowych i wewnątrzkomórkowych z hodowli astrocytów