This work was supported by research grants DK52057 and DK107498 from the NIH to K.V.K. X.P was supported by the institutional training grant 2T32DK007201 from the NIH.

1. Handling of the Peptide
<ol>
	<li>Design and ordering of the peptide
	<ol>
		<li>Choose the desired sequence of the peptide, and add a tag, such as myc epitope (EQKLISEED) at its either N- or C-terminus.</li>
		<li>Check the predicted solubility of the peptide in water, using peptide solubility calculator http://pepcalc.com/peptide-solubility-calculator.php. If the solubility is low, try to add another water-soluble tag to change the charge balance. 
		NOTE: We used the peptide corresponding to the first luminal loop (fll) of GLUT4 tagged with the myc epitope (bold font) at the N terminus (Myc-fll-GLUT4): EQKLISEEDLNAPQKVIEQSYNATWLGRQGPGGPSSIPPGTLTTLWA that has &#34;good&#34; predicted solubility in water.</li>
		<li>Order the custom peptide with at least 75% purity which is sufficient for the assay, and ask the provider for solubility test. Separate the order into at least two aliquots in case of unforeseen problems with dissolving the peptide. 
		NOTE: Myc-fll-GLUT4 was ordered in two aliquots. Its reported solubility in ultrapure water is &#8804;10 mg/mL.</li>
	</ol>
	</li>
	<li>Dissolving the peptide 
	Note: Ultrapure water is the best solvent. If the peptide does not appear to be water soluble, refer to http://www.genscript.com/peptide_solubility_and_stablity.html for instructions.
	<ol>
		<li>Prepare 100x working solution of the peptide in ultrapure water or in buffer of choice, with the concentration of 100 &#956;g/mL.</li>
		<li>Separate the solution into 50 - 100 &#181;L aliquots, and store them at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Handling of Cells
<ol>
	<li>Cell culture
	<ol>
		<li>Use Wild type (WT) cells as a negative control, and cells expressing the target protein tagged with six histidines (HisP). 
		NOTE: We used 3T3 L1 pre-adipocytes stably transfected with Sortilin-myc/His5.</li>
		<li>Grow the cells in Dulbecco&#39;s Modified Eagle Medium (DMEM) high Sugar, supplemented with 10% calf bovine serum, glutamine (2 mM) and penicillin/streptomycin (5 &#956;g/mL) at 37 &#176;C in 10% CO2.</li>
		<li>Sit four 10 cm dishes of cells, transfect two of them with HisP and transfect the other two with control plasmid (WT). 48 h after transfection, cells should reach 80 - 90% confluency.</li>
	</ol>
	</li>
	<li>Preparation of the Cell Lysates
	<ol>
		<li>Prepare lysis buffer (10 mM Hepes, 30 mM NaCl, 5% glycerol, 10 mM Imidazole, 0.5% Triton X-100 and protease inhibitor cocktail without EDTA for the isolation of His-tagged proteins, pH 7.4) and keep it at 4 &#176;C:</li>
		<li>Wash cells three times with 10 mL of 1x phosphate-buffered saline (PBS) at 4 &#176;C.</li>
		<li>Put the dishes on ice and add 500 &#181;L of lysis buffer to each dish. Harvest the cell lysates using a cell scraper.</li>
		<li>Place the cell lysate from each dish into a different 1.5 mL tube. Label the tubes as WT-pH6, WT-pH8, HisP-pH6, HisP-pH8.</li>
		<li>Pass the cell lysates through a syringe with a 26G needle five times up and down, to complete lysis.</li>
		<li>Centrifuge the cell lysates at 16,000 x g for 10 min at 4 &#176;C.</li>
		<li>Transfer the supernatants to new identically labeled tubes.</li>
		<li>Analyze protein concentration using the BCA Protein Assay Kit or other kit.</li>
		<li>Using lysis buffer, equalize protein concentration of all four lysates.</li>
		<li>Separate a small (ca. 20 &#181;L) aliquot of each lysate and keep it at -20 &#176;C for possible control experiments and/or trouble shooting.</li>
	</ol>
	</li>
</ol>
3. Binding of His-tagged Proteins to the HisPur Cobalt Beads
<ol>
	<li>Use commercially available wash buffer or prepare one (50 mM Na2HPO4/NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8) and keep it at 4 &#176;C.</li>
	<li>Prepare wash buffer with pH 6 by tittering wash buffer pH 8 with HCl. Keep it at 4 &#176;C.</li>
	<li>Gently swirl the bottle of the cobalt beads to make homogenous suspension.</li>
	<li>Dispense 40 &#181;L of the Cobalt beads suspension into four 1.5 mL tubes marked as above (step 2.2.4).</li>
	<li>Add 1 mL of lysis buffer to each tube, and centrifuge the tubes at 1000 x g for 5 s. Aspirate the supernatant, and add 40 &#956;L of lysis buffer to settled beads.</li>
	<li>Add the cell lysates to corresponding tubes.</li>
	<li>Incubate the tubes for 90 min at 4 &#176;C on a tube rotator at 20 rpm.</li>
	<li>Centrifuge the tubes at 1000 x g for 5 s and collect the supernatant. Keep the supernatant at 4 &#176;C.</li>
	<li>Add 500 &#181;L of either pH 8 or pH 6 wash buffer to corresponding tubes and re-suspend the beads gently.</li>
	<li>Centrifuge the tubes at 1000 x g for 5 s and discard the supernatants.</li>
	<li>Repeat steps 3.9 and 3.10 for four times.</li>
</ol>
4. Binding of the Peptide to the His-tagged Protein Immobilized on the Beads
<ol>
	<li>Using 100x working solution of the peptide (step 1.2.1), prepare 1x working solutions in either pH 6 or pH 8 wash buffer (two 100 &#181;L aliquots for each, 1 &#181;g/mL).</li>
	<li>Add 100 &#956;L of 1x peptide solution to the washed beads with the corresponding pH.</li>
	<li>Incubate the beads for 30 min at 4 &#176;C on a tube rotator at 20 rpm.</li>
	<li>Centrifuge the tubes at 1,000 x g for 5 s, collect the supernatant and keep it at -20 &#176;C.</li>
	<li>Repeat steps 3.9 and 3.10 for four times. 
	NOTE: In order to decrease possible background, the following steps could replace steps 4.4 &#38; 4.5.</li>
	<li>Take four micro columns with 30 &#956;m pores, label them as in step 2.2.4, and place the columns in collection tubes.</li>
	<li>After completion of step 4.3, transfer the incubation mixture into the columns, allow the solution to pass through by gravity. Keep the flow through at -20 &#176;C.</li>
	<li>Pass 500 &#181;L of wash buffer with pH 6 or pH 8 through corresponding columns by gravity. Discard the flow through.</li>
	<li>Repeat step 4.8 four times.</li>
	<li>After the last wash, centrifuge the columns at 1,000 x g for 15 s.</li>
</ol>
5. Elution from Cobalt Beads
<ol>
	<li>Use commercial Tricine sample buffer (200 mM Tris-HCl, 40% glycerol, 2% SDS, 0.04% Coomassie Blue, pH 6.8) and add &#946;-mercaptoethanol to the final concentration of 2%.</li>
	<li>Add 40 &#181;L of Tricine sample buffer (with &#946;-mercaptoethanol) to the washed beads and vortex the sample.</li>
	<li>Heat the samples for 10 min at 100 &#176;C, vortex the samples again, and centrifuge the tubes at 1,000 x g for 5 s. 
	NOTE: In order to decrease possible nonspecific binding in the elution, the following steps could replace steps 5.1 - 5.3.</li>
	<li>Add 40 &#956;L of Elution Imidazole Buffer (50 mM Na2HPO4/NaH2PO4, 300 mM NaCl, 0.25 M imidazole) to the washed beads, vortex and incubate the tubes at room temperature for 20 min.</li>
	<li>Centrifuge the tubes at 3,000 x g for 30 s, collect the supernatant (eluted samples) and transfer it to new labeled tubes.</li>
	<li>Add 40 &#956;L of Tricine sample buffer to each tube, heat the samples for 10 min at 100 &#176;C, vortex and centrifuge the tubes at 1,000 x g for 5 s. 
	NOTE: If micro columns are used, add the elution buffer to the washed beads, incubate for 20 min&#160;and collect the elution by centrifugation at 8,000 x g for 2 min. 
	NOTE: Samples can be kept at -20 &#176;C till electrophoresis is performed.</li>
</ol>
6. Electrophoresis and Western blotting
Note: The samples are ready for the separation by SDS-PAGE and subsequent Western blotting. Follow protocol of gel electrophoresis (https://www-jove-com.remotexs.ntu.edu.sg/science-education/5065/the-western-blot) with the following modifications.
<ol>
	<li>Use commercially available 10 - 20% Tricine gradient gels.</li>
	<li>On the gel, load 20 &#181;L of the eluted samples and 20 &#181;L of lysates (step 2.2.10). For trouble shooting, it is recommended to load 20 &#181;L of unbound material (step 3.8) and 10 ng of the peptide; before loading, add equal amount of Tricine sample buffer to these samples.</li>
	<li>Carry out electrophoresis in commercial Tris/Tricine/SDS running buffer (100 mM Tris, 100 mM Tricine, and 0.1% SDS, pH 8.3).</li>
	<li>Transfer the material from the gel onto a 0.45 &#181;m nitrocellulose membrane using transfer buffer (25 mM Tris base, 192 mM glycine, pH 8.4) supplemented with 20% methanol.</li>
	<li>Block the membrane with 3% Bovine Serum Albumin (BSA) for 1 h at room temperature. Using pre-stained molecular weight markers as guides, cut the membrane horizontally, blot the top part with an antibody against HisP, and blot the bottom part with an antibody against the myc epitope to identify the myc-tagged peptide. 
	NOTE: In our particular case, cutting of the membrane is not required since both HisP and Myc-fll-GLUT4 can be detected with an anti-myc antibody.</li>
</ol>
7. Analysis of Results
<ol>
	<li>Quantify the intensity of all Western blot signals using an Image Station or ImageJ program.</li>
	<li>Normalize the signal from the myc-tagged peptide against the signal from HisP at both pH values.</li>
</ol>

<ol>
	<li>Bogan, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+glucose+transporter+translocation+in+health+and+diabetes.">Regulation of glucose transporter translocation in health and diabetes.</a> Annu Rev Biochem. 81, 507-532 (2012).</li><li>Kandror, K. V., Pilch, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sugar+is+sIRVed:+sorting+Glut4+and+its+fellow+travelers.">The sugar is sIRVed: sorting Glut4 and its fellow travelers.</a> Traffic. 12 (6), 665-671 (2011).</li><li>Pan, X., Zaarur, N., Singh, M., Morin, P., Kandror, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sortilin+and+retromer+mediate+retrograde+transport+of+Glut4+in+3T3-L1+adipocytes.">Sortilin and retromer mediate retrograde transport of Glut4 in 3T3-L1 adipocytes.</a> Mol Biol Cell. 28 (12), 1667-1675 (2017).</li><li>Kim, J., Kandror, K. 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+first+luminal+loop+confers+insulin+responsiveness+to+the+glucose+transporter+4.">The first luminal loop confers insulin responsiveness to the glucose transporter 4.</a> Mol Biol Cell. 23 (5), 910-917 (2012).</li><li>Shi, J., Kandror, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sortilin+is+essential+and+sufficient+for+the+formation+of+Glut4-storage+vesicles+in+3T3-L1+adipocytes.">Sortilin is essential and sufficient for the formation of Glut4-storage vesicles in 3T3-L1 adipocytes.</a> Dev Cell. 9 (1), 99-108 (2005).</li><li>Coutinho, M. F., Prata, M. J., Alves, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+shortcut+to+the+lysosome:+the+mannose-6-phosphate-independent+pathway.">A shortcut to the lysosome: the mannose-6-phosphate-independent pathway.</a> Mol Genet Metab. 107 (3), 257-266 (2012).</li><li>Willnow, T. E., Petersen, C. M., Nykjaer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VPS10P-domain+receptors+-+regulators+of+neuronal+viability+and+function.">VPS10P-domain receptors - regulators of neuronal viability and function.</a> Nat Rev Neurosci. 9 (12), 899-909 (2008).</li><li>Mazella, J., 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=The+100-kDa+neurotensin+receptor+is+gp95/sortilin,+a+non-G-protein-coupled+receptor.">The 100-kDa neurotensin receptor is gp95/sortilin, a non-G-protein-coupled receptor.</a> J Biol Chem. 273 (41), 26273-26276 (1998).</li><li>Ni, X., Morales, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lysosomal+Trafficking+of+Acid+Sphingomyelinase+is+Mediated+by+Sortilin+and+Mannose+6-phosphate+Receptor.">The Lysosomal Trafficking of Acid Sphingomyelinase is Mediated by Sortilin and Mannose 6-phosphate Receptor.</a> Traffic. 7 (7), 889-902 (2006).</li><li>Westergaard, U. B., 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=Functional+organization+of+the+sortilin+Vps10p+domain.">Functional organization of the sortilin Vps10p domain.</a> J Biol Chem. 279 (48), 50221-50229 (2004).</li><li>Nykjaer, A., 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=Sortilin+is+essential+for+proNGF-induced+neuronal+cell+death.">Sortilin is essential for proNGF-induced neuronal cell death.</a> Nature. 427 (6977), 843-848 (2004).</li></ol>

The authors have nothing to disclose.

Sortilin is an evolutionary conserved multi-ligand protein receptor that is involved in both signaling at the plasma membrane and in intracellular sorting events6,7. However, the search for the authentic sortilin&#39;s ligands (some of which are luminal or integral membrane proteins) is complicated as the interaction of sortilin with some of its binding partners appears to be sensitive to detergents. Therefore, an easy and a widespread approach for studying protein-protein interactions, co-immunoprecipitation, cannot be readily applied. Some research groups have used co-immunoprecipitation to demonstrate the interaction between sortilin and its potential ligands8,9. However, these experiments have been carried out either in 0.1% Triton or in 0.6% CHAPS which casts doubts in the completeness of membrane solubilization.
Other researchers studied the interaction between sortilin and its ligands by surface plasmon resonance10,11. Although surface plasmon resonance is arguably the best approach to the problem, it requires expensive equipment and significant amounts of pure recombinant proteins which is costly and time consuming.
Here, we describe a technique that, in combination with other methods, such as cross-linking, and the yeast two hybrid system, strongly suggest that sortilin can bind to GLUT4. The assay is fast and easy and can be readily adapted to other proteins and different sizes of peptides.
There are a few critical steps in this assay. The first one is the peptide&#39;s solubility in water. Another one is the selection of the proper control. Clearly, a significant amount of the biological material can interact with cobalt beads via endogenous His-reach sequences or non-specifically. Therefore, it is essential to immobilize on the beads lysates of WT cells and cells transfected with the protein of interest, in our case Sortilin-myc/His. In such an experimental design, material bound to the beads will differ in only one protein, Sortilin-myc/His, so that a certain degree of background binding of the peptide to control beads can be tolerated. Still, the background binding of the peptide to the beads can be reduced by increasing the stringency of the final washing steps. The use of mini columns for washing the beads may decrease background binding even further.
Because of the nature of the &#34;GLUT4 pathway&#34;, we wanted to address binding of Myc-fll-GLUT4 to sortilin at different pH values. We realize that pH in the lumen of early/sorting endosomes can fall below 6. However, pH lower than 6 disrupts binding of His-tagged proteins to cobalt beads which may be considered as a limitation of the method.
Isolation of the target protein on cobalt beads using the His epitope is preferable to other affinity purification steps, such as immunoprecipitation with specific antibodies, in terms of the yield and non-specific binding. Still, it is completely possible that isolation of the target protein on any other resin (preferably, magnetic beads) with proper controls will prove successful.

GLUT4 is a glucose transporter protein which is expressed predominantly in fat and skeletal muscle cells where it mediates the effect of insulin on post-prandial blood glucose clearance1. Being a very stable protein, GLUT4 is regulated at a post-translational level. In the absence of insulin, GLUT4 is largely excluded from the plasma membrane (hence low basal permeability for glucose) and is localized mainly inside the cell in small insulin-responsive vesicles (IRVs) and trans-Golgi network (TGN) that is likely to represent the IRV donor compartment. Upon insulin administration, the IRVs fuse with the plasma membrane and deliver GLUT4 to the site of its functioning. This increases the permeability of the plasma membrane for glucose, so that glucose uptake from blood into adipocytes and skeletal myocytes rises 10 to 40-fold. After insulin withdrawal, GLUT4 is internalized into early/sorting endosomes and then retrieved to TGN where the IRVs are re-formed. Both sorting steps in the GLUT4 pathway, i.e. retrieval from the peripheral early endosomes to the perinuclear TGN, and the formation of the IRVs on the TGN donor membranes are enabled by the Vps10p family member, sortilin, which represents a type I transmembrane protein and a sorting receptor. According to one model, sortilin works as a transmembrane scaffold protein: it binds GLUT4 in the lumen of endosomes and TGN, and recruits retromer or clathrin adaptors to the cytoplasmic side of the donor membrane via its C-terminus2,3. This facilitates the distribution of GLUT4 into vesicular carriers that translocate GLUT4 between intracellular compartments.
The interaction of the cytoplasmic tail of sortilin with retromer and various adaptor proteins has been well documented. However, the binding of sortilin to GLUT4 (and to several of its other protein ligands) has been challenging to prove. In particular, attempts to co-immunoprecipitate sortilin and GLUT4 have not been successful probably due to the detergent-sensitive nature of the interaction between these two proteins. In addition, as a typical transporter protein, GLUT4 has 12 transmembrane domains and 6 luminal loops any combination of which may potentially represent a sortilin-binding site. At the same time, a large body of indirect evidence, such as substantial co-localization in the cell, cross-linking with membrane-permeable DSP, and the interaction in yeast two hybrid system suggest that sortilin can bind to GLUT4. Furthermore, using the latter approach in a combination with the alanine scanning mutagenesis, we have previously determined that the Vps10p domain of sortilin binds primarily to the first luminal loop of GLUT4. However, the proof of such an interaction in mammalian cells has been missing. Here, we have isolated His-tagged sortilin from transfected 3T3-L1 cells using cobalt resin and demonstrated that it can interact with chemically synthesized peptide corresponding to the first luminal loop of GLUT4 at pH 6 and pH 8 that resemble acidic milieu in the endosomal lumen and neutral environment in the lumen of the TGN membranes. No peptide binding was detected in control experiments where extract prepared from non-transfected cells was loaded on the same beads.

<table border="0" cellpadding="0" cellspacing="0" width="608">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protease inhibitor cocktail</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">P8849</td>
			<td style="width:167px;">for use in purification of histidine tag protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosphate-Buffered Saline&#160;</td>
			<td style="width:107px;">Corning</td>
			<td style="width:119px;">21-040-CV</td>
			<td style="width:167px;">PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1mL Insulin Syringe U-100</td>
			<td style="width:107px;">BD</td>
			<td style="width:119px;">329652</td>
			<td style="width:167px;">26G x 1/2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BCA Protein Assay Kit</td>
			<td style="width:107px;">Pierce</td>
			<td style="width:119px;">23228</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Wash buffer</td>
			<td style="width:107px;">Boston BioProducts</td>
			<td style="width:119px;">BP-234</td>
			<td style="width:167px;">For His-tag protein purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HisPur Cobalt Resin</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:119px;">89964</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Tricine sample Buffer</td>
			<td style="width:107px;">Bio-Rad</td>
			<td style="width:119px;">161-0739</td>
			<td style="width:167px;">with SDS, bMercaptaethanol should be added</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Elution&#160; Buffer</td>
			<td style="width:107px;">Boston BioProducts</td>
			<td style="width:119px;">BP-236</td>
			<td style="width:167px;">For His-tag protein purificationwith 250mM Imidazole</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Mini-Protean Tris-Tricine precast gels 10-20%&#160;</td>
			<td style="width:107px;">Bio-Rad</td>
			<td style="width:119px;">456-3115</td>
			<td style="width:167px;">For seperation of peptide and small protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris/Tricine/SDS running buffer&#160;</td>
			<td style="width:107px;">Bio-Rad</td>
			<td style="width:119px;">161-0744</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Transfer Buffer</td>
			<td style="width:107px;">Boston BioProducts</td>
			<td style="width:119px;">BP-190</td>
			<td style="width:167px;">Add 20% methanol</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nitrocellulose membrane&#160; 0.45 mm</td>
			<td style="width:107px;">Bio-Rad</td>
			<td style="width:119px;">1620115</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">A9647</td>
			<td style="width:167px;">BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti Myc antibody</td>
			<td style="width:107px;">Cell Signaling</td>
			<td style="width:119px;">2272</td>
			<td style="width:167px;">Rabbit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Peptide</td>
			<td style="width:107px;">Genscipt</td>
			<td style="width:119px;"></td>
			<td>customize ordering</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Precision Plus Protein Dual color Standarts</td>
			<td style="width:107px;">BioRad</td>
			<td style="width:119px;">161-0374</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of detergent-sensitive interactions between membrane proteins

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in many cases is associated with significant experimental challenges. In particular, sorting receptors interact with their protein cargo in the lumen of the membrane compartments often in a detergent-sensitive fashion, making co-immunoprecipitation of these proteins unusable. Binding of the sorting receptor sortilin to glucose transporter GLUT4 may serve as an example of weak luminal interactions between membrane proteins. Here, we describe a fast, simple, and inexpensive assay to validate the interaction between sortilin and GLUT4. For that, we have designed and chemically synthesized the myc-tagged peptide corresponding to the potential sortilin-binding epitope in the luminal part of GLUT4. Sortilin tagged with six histidines was expressed in mammalian cells, and isolated from cell lysates using Cobalt beads. Sortilin immobilized on the beads was incubated with the peptide solution at different pH values, and the eluted material was analyzed by Western blotting. This assay can be easily adapted to study other detergent-sensitive protein-protein interactions.

Lysates were prepared from 3T3 L1 cells stably transfected with Sortilin-myc/His5 and from WT 3T3 L1 cells, used as a negative control. Both lysates were incubated with cobalt beads at pH 6 or pH 8 and thoroughly washed. Beads with immobilized proteins were then incubated in the solution of Myc-fll-Glut4. After careful washes, proteins bound to the beads were eluted with 0.25 M Imidazole. Samples were subjected, along with the original lysates, to SDS-PAGE in a 10-20% Tricine gradient gel followed by Western blotting with anti-Myc antibody that allowed for the detection of both sortilin-myc/His (110 kD) and Myc-fll-GLUT4 (5 kD) (Figure 1).
Myc-fll-GLUT4 (10 ng) was loaded on the first lane of the gel as a reference. As the purity of the peptide is only 75%, contaminations are apparent (higher bands). Original cell lysates from both WT and Sortilin-myc/His expressing cells contain multiple bands recognized by the anti-myc antibody. Material isolated from Sortilin-myc/His expressing cells is much cleaner. In addition to some minor non-specific bands, it has only two myc-containing components: Sortilin-myc/His and Myc-fll-GLUT4. Contaminating peptides in the first lane do not appear to bind to Sortilin-myc/His. Negative control (WT eluate) has predominantly non-specific bands.
As GLUT4 passes through intracellular compartments, such as endosomes and TGN, that have different luminal pH, we wanted to assess interaction of GLUT4 with sortilin at pH 6 and pH 8 (Figure 2). Densitometry of results (not shown) has not revealed any significant differences in the ratio between Myc-fll-GLUT4 and Sortilin-myc/His retained on the beads at different pH values. We thus conclude that GLUT4 has the ability to interact with sortilin in both endosomes and TGN.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57179/57179fig1v2.jpg" /> 
Figure 1. Interaction of Sortilin-myc/His with Myc-fll-GLUT4 on cobalt beads. Lysates prepared from WT 3T3-L1 cells and 3T3-L1 cells stably expressing Sortilin-myc/His (S) were passed over cobalt beads, washed, incubated with Myc-fll-Glut4 at pH 8, washed again, and eluted with Imidazole buffer. Myc-fll-Glut4 (10 ng) was loaded on the first lane as a reference.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57179/57179fig2v2.jpg" /> 
Figure 2. Sortilin-myc/His interacts with Myc-fll-GLUT4 at both pH 8 and pH 6. Myc-fll-Glut4 was incubated with material immobilized on the beads at pH 6 and pH 8, washed, and eluted with Glycine sample buffer. The figure has been adapted from Pan X., et al3.

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Detection of Detergent-sensitive Interactions Between Membrane Proteins

We describe a protocol for detection of detergent-sensitive interactions between membrane proteins using binding of the sorting receptor, sortilin, to the first luminal loop of the glucose transporter protein, GLUT4, as an example.

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

detection-detergent-sensitive-interactions-between-membrane

Nanyang Technological University

Research

JoVE Journal

Biochemistry

5.9K Views.  Boston University School of Medicine. We describe a protocol for detection of detergent-sensitive interactions between membrane proteins using binding of the sorting receptor, sortilin, to the first luminal loop of the glucose transporter protein, GLUT4, as an example. 

Video: Detection of Detergent-sensitive Interactions Between Membrane Proteins

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs

Cell-free expression systems allow the tailored design of reaction environments to support the functional folding of even complex proteins such as membrane proteins. The experimental procedures for the co-translational insertion and folding of membrane proteins into preformed and defined membranes supplied as nanodiscs are demonstrated. The protocol is completely detergent-free and can generate milligrams of purified samples within one day. The resulting membrane protein/nanodisc samples can be used for a variety of functional studies and structural applications such as crystallization, nuclear magnetic resonance, or electron microscopy. The preparation of basic key components such as cell-free lysates, nanodiscs with designed membranes, critical stock solutions as well as the assembly of two-compartment cell-free expression reactions is described. Since folding requirements of membrane proteins can be highly diverse, a major focus of this protocol is the modulation of parameters and reaction steps important for sample quality such as critical basic reaction compounds, membrane composition of nanodiscs, redox and chaperone environment, or DNA template design. The whole process is demonstrated with the synthesis of proteorhodopsin and a G-protein coupled receptor.

Co-translational insertion into pre-formed nanodiscs makes it possible to study cell-free synthesized membrane proteins in defined lipid environments without contact with detergents. This protocol describes the preparation of essential system components and the critical parameters for improving expression efficiency and sample quality.