We thank Professor Dontscho Kerjaschki, Corina Mayrhofer and Sigurd Krieger at the Institute of Clinical Pathology, University of Vienna, Austria for their collaboration.

Cell culture
<ol>
<li>Grow Chinese Hamster Ovary cells (CHO-K1) using standard cell culture procedures in F-12 Ham medium with GlutaMAX I containing 10% fetal calf serum, 50 U/ml penicillin, and 50 &micro;g/ml streptomycin sulfate (Invitrogen).</li>
<li>Exchange the culture medium to serum-free media. Label the cell surface proteins were at different time points with CyDye DIGE Fluor Cy3 or Cy5 minimal dyes (see section Cell Surface Labeling, below).</li>
<li>Pool equal numbers of cells from each time point and label with CyDye DIGE Fluor Cy2. Use&nbsp;these as an internal standard for each 2-D gel.</li>
<li>The majority of the experiments can be performed with CHO-K1 cells, but mouse embryo fibroblasts (3T3 L1) and mouse ascites lymphoma lymphoblasts (EL4) can also used (data not shown).</li>
<li>Grow the two latter cell types in DMEM medium with GlutaMAX II, but, otherwise, use&nbsp;identical conditions used for the CHO-K1 cells.</li>
</ol>
Cell surface labeling
<ol>
<li>Carefully detach adherent cells non-enzymatically, counting and dividing into aliquots of 5&ndash;10 x106 cells. For cells growing in suspension, omit the detaching step.</li>
<li>Centrifuge the cell suspensions at about 800 x g for 5 minutes. Remove&nbsp;the supernatants containing the medium.</li>
<li>Wash the pellets by resuspendion in 1 ml ice cold Hank&rsquo;s Balanced Salt Solution (HBSS) pH 8.5.&nbsp;Centrifuged at 800 x g at 4&deg;C for 2 minutes.</li>
<li>Remove&nbsp;the supernatant and resuspend the cell pellet in 200 &micro;l ice cold labeling buffer (HBSS pH 8.5, 1 M urea).</li>
<li>Label the intact cells with 600 pmol CyDye DIGE Fluor minimal dyes for 20 minutes on ice in the dark.</li>
<li>Quench the reaction by adding 20 &micro;l 10 mM lysine. Incubate for 10 minutes.</li>
<li>Wash the surface-labeled cells twice by resuspension in 500 &micro;l HBSS pH 7.4,&nbsp;followed by centrifugation at 800 x g at 4&deg;C for 2 minutes.</li>
</ol>
Cell lysis and fractionation
<ol>
<li>Lyse the surface-labeled cells in 150 &mu;l cold lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris, 5 mM magnesium acetate pH 8.5). Leave on ice for at least 1 h with occasional vortexing.</li>
<li>Centrifuge the lysates at 10 000 x g at 4&deg;C for 5 minutes. Transfer&nbsp;the supernatant to a new tube. This sample is the non-fractionated sample containing all cellular proteins.</li>
<li>In parallel, wash&nbsp;cell pellets, as above, then fractionate (using a membrane fractionation kit, Pierce) into membrane and cytosolic fractions prior to 2-D gel electrophoresis. The membrane fraction contains internal and cell surface membrane proteins.</li>
<li>For comparison, follow the standard Ettan DIGE procedure 3, and lyse, label, and, finally, fractionate the cells. Determine the protein concentration in the samples using the 2- D Quant Kit (GE Healthcare).</li>
</ol>
2-D electrophoresis
<ol>
<li>Rehydrate Immobiline DryStrip gels, pH 3&ndash;11NL (24 cm) using Immobiline DryStrip Reswelling tray, 24 cm in 450 &micro;l DeStreak Rehydration solution (0.5% IPG Buffer) overnight.</li>
<li>Apply the CyDye-labeled samples (corresponding to 50 &micro;g total protein) to Immobiline DryStrip gels by anodic cup loading in the manifold and perform isoelectric focusing (IEF) using Ettan IPGphor II IEF System according to instructions.</li>
<li>After IEF, equilibrate the strips in two steps and place on top of large (26 x 20 cm) 12.5% polyacrylamide gels (SDS-PAGE). Overlay with 0.5% agarose (in running buffer containing bromophenol blue). Run 2-D electrophoresis using Ettan DALTtwelve Large Vertical System at 5 W/gel for 30 min, and then at 15 W/gel until the dye front reaches the bottom of the gel.</li>
</ol>
Imaging and data analysis
<ol>
<li>After completing 2-D electrophoresis, scan the gels for Cy2, Cy3 or Cy5 using a Typhoon 9410 Variable Mode Imager.</li>
<li>Compare spot maps&nbsp;from membrane fractions, cytosolic fractions, and non-fractionated samples using DeCyder 2-D Differential Analysis Software 4.</li>
</ol>
Post-staining
<ol>
<li>After imaging, silver stain the gels according to standard procedure 5.</li>
</ol>
Protein identification
<ol>
<li>Grow, harvest, wash and lyse preparative amounts (approximately 1 mg total protein from 10 x106 CHO cells) of cells, as described above for a non-fractionated sample. Use&nbsp;a Cy5 cell surface labeled sample (see above) as a spike, and apply together with the unlabelled preparative amounts of protein.</li>
<li>Carry out 2-D electrophoresis as described above, but this time, aplly 600 &micro;g unlabeled cell lysate together with 50 &micro;g cell surface labeled spike by anodic cup application. Use reference markers to allow correct spot picking, and place between the glass plates before gel casting according to recommendations 3.</li>
<li>Scan the gel in Cy5 channel to obtain the Cy5 cell surface spot map, followed by total protein staining using Deep purple total protein stain 3.</li>
<li>Match together the two spot maps using the DeCyder 2-D software and create a pick list for all the spots corresponding to the Cy 5 labeled cell surface proteins. Pick cell surface proteins using the Ettan spot handling station, extract from the gel plugs, and trypsinate using the Ettan digester. Identify using MALDI-TOF mass spectrometry.</li>
</ol>

Table 1. An Ettan DIGE experiment was performed using samples from serum depleted cells labeled according to the cell-surface protein labeling protocol in figure 1.

<table style="width: 517px; height: 139px;" border="1" cellspacing="0" cellpadding="0">
<tbody>
<tr>
<td style="text-align:center; font-weight:bold">Sample</td>
<td style="text-align:center; font-weight:bold">Time of serum depletion</td>
<td style="text-align:center; font-weight:bold">Labelled with CyDye</td>
<td style="text-align:center; font-weight:bold">Gel&nbsp; number</td>
</tr>
<tr>
<td style="text-align:center;">1</td>
<td style="text-align:center;">-</td>
<td style="text-align:center;">Cy 3, Cy 2</td>
<td style="text-align:center;">1</td>
</tr>
<tr>
<td style="text-align:center;">2</td>
<td style="text-align:center;">30 minutes</td>
<td style="text-align:center;">Cy 5, Cy 2</td>
<td style="text-align:center;">1</td>
</tr>
<tr>
<td style="text-align:center;">3</td>
<td style="text-align:center;">2 hours</td>
<td style="text-align:center;">Cy 3, Cy 2</td>
<td style="text-align:center;">2</td>
</tr>
<tr>
<td style="text-align:center;">4</td>
<td style="text-align:center;">4 hours</td>
<td style="text-align:center;">Cy 5, Cy 2</td>
<td style="text-align:center;">2</td>
</tr>
<tr>
<td style="text-align:center;">5</td>
<td style="text-align:center;">16 hours</td>
<td style="text-align:center;">Cy 5, Cy 2</td>
<td style="text-align:center;">3</td>
</tr>
</tbody>
</table>

&nbsp;

<ol>
	<li>Jang, J. H., Hanash, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+the+cell+surface+proteome.">Profiling of the cell surface proteome.</a> Proteomics. 3, 1947-1954 (2003).</li><li>Shin, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+profiling+of+the+cell+surface+proteome+of+cancer+cells+uncovers+an+abundance+of+proteins+with+chaperone+function.">Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function.</a> J Biol Chem. 9, 7607-7616 (2003).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ettan+DIGE+System+User+Manual.">Ettan DIGE System User Manual.</a> GE Healthcare user manual 18-1164-40. , (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DeCyder+2-D+Differential+Analysis+Software+v+6.0.+GE+Healthcare+data+file+11-0011-98+Edition+AA.">DeCyder 2-D Differential Analysis Software v 6.0. GE Healthcare data file 11-0011-98 Edition AA.</a> , (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2-D+Electrophoresis+using+immobilized+pH+gradients,+principles+and+methods.">2-D Electrophoresis using immobilized pH gradients, principles and methods.</a> GE Healthcare handbook 80-6429-60 Edition AB. , (2002).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CyDye+DIGE+fluors+and+labeling+kits.+GE+Healthcare+data+file+18-1164-84+Edition+AB.">CyDye DIGE fluors and labeling kits. GE Healthcare data file 18-1164-84 Edition AB.</a> , (2003).</li><li>Mayrhofer, C., Krieger, S., Allmaier, G., Kerjaschki, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DIGE+compatible+labeling+of+surface+proteins+on+vital+cells+in+vitro+and+in+vivo.">DIGE compatible labeling of surface proteins on vital cells in vitro and in vivo.</a> Proteomics. 2, 579-5785 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+labeling+of+cell-surface+proteins+using+CyDye+DIGE+Fluor+minimal+dyes.+GE+Healthcare+Application+Note+11-0033-92+Edition+AB.">Selective labeling of cell-surface proteins using CyDye DIGE Fluor minimal dyes. GE Healthcare Application Note 11-0033-92 Edition AB.</a> , (2005).</li></ol>

Protein concentration
An overview of the two labeling workflows is shown in Figure 1. Since the cells are still intact when labeled according to the cell-surface protein labeling protocol, only the cell surface proteins are exposed to the dye. In the standard Ettan DIGE protocol, the cells are lysed before labeling and proteins inside as well as outside the cell are labeled (Fig 1). The relative amount of dye to protein in the cell-surface protein labeling protocol is not known, since cell-surfa...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CyDye DIGE Kit, 2 nmol</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>28-9345-30</td>
<td>&nbsp;</td>
<tr>
<td>2-D Quant Kit</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>80-6484-51</td>
<td>&nbsp;</td>
<tr>
<td>IPG Buffer pH 3-11NL</td>
<td>Reagent</td>
<td>GE Heallthcare</td>
<td>17-6004-40</td>
<td>&nbsp;</td>
<tr>
<td>Immobiline DryStrip pH 3-11NL, 24 cm</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-6003-77</td>
<td>&nbsp;</td>
<tr>
<td>IPGbox</td>
<td>Tool</td>
<td>GE Healthcare</td>
<td>28-9334-65</td>
<td>&nbsp;</td>
<tr>
<td>IPGbox kit</td>
<td>Tool</td>
<td>GE Healthcare</td>
<td>28-9334-92</td>
<td>&nbsp;</td>
<tr>
<td>DeStreak Rehydration solution</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-6003-19</td>
<td>&nbsp;</td>
<tr>
<td>Immobiline DryStrip Cover Fluid</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1335-01</td>
<td>&nbsp;</td>
<tr>
<td>Ettan IPGphor 3 IEF Unit</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>11-0033-64</td>
<td>&nbsp;</td>
<tr>
<td>Ettan IPGphor Manifold</td>
<td>Instrument component</td>
<td>GE Healthcare</td>
<td>80-6498-38</td>
<td>&nbsp;</td>
<tr>
<td>Ettan DALTtwelve Gel Caster </td>
<td>Tool</td>
<td>GE Healthcare</td>
<td>80-6467-22</td>
<td>&nbsp;</td>
<tr>
<td>Ettan DALTtwelve Separation Unit and Power Supply/Control Unit</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>80-6466-46</td>
<td>230 V unit: 80-6466-27</td>
</tr>
<tr>
<td>Typhoon 9410 Variable Mode Imager</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>63-0055-81</td>
<td>&nbsp;</td>
<tr>
<td>Ettan Spot Picker</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>18-1145-28</td>
<td>&nbsp;</td>
<tr>
<td>Ettan Digester</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>18-1142-68</td>
<td>&nbsp;</td>
<tr>
<td>Ettan Spotter</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>18-1142-67</td>
<td>&nbsp;</td>
<tr>
<td>Ettan MALDI-TOF</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>18-1142-33</td>
<td>&nbsp;</td>
<tr>
<td>DeCyder 2-D Differential Analysis Software</td>
<td>Other</td>
<td>GE Healthcare</td>
<td>28-4012-01</td>
<td>&nbsp;</td>
<tr>
<td>ImageQuant Analysis Software</td>
<td>Other</td>
<td>GE Healthcare</td>
<td>28-9236-62</td>
<td>&nbsp;</td>
<tr>
<td>Urea</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1319-01</td>
<td>&nbsp;</td>
<tr>
<td>CHAPS</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1314-01</td>
<td>&nbsp;</td>
<tr>
<td>PlusOne Dithiothreitol (DTT)</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1318-01</td>
<td>&nbsp;</td>
<tr>
<td>Bromophenol Blue</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1329-01</td>
<td>&nbsp;</td>
<tr>
<td>Tris</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1321-01</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Dodecylsulfate (SDS)</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1313-01</td>
<td>&nbsp;</td>
<tr>
<td>PlusOne Glycerol 87%</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1325-01</td>
<td>&nbsp;</td>
<tr>
<td>PlusOne ReadySol IEF 40% T, 3% C</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1310-01</td>
<td>&nbsp;</td>
<tr>
<td>PlusOne TEMED</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1312-01</td>
<td>&nbsp;</td>
<tr>
<td>PlusOne Ammonium Persulfate</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1311-01</td>
<td>&nbsp;</td>
<tr>
<td>PlusOne Glycine </td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1323-01</td>
<td>&nbsp;</td>
<tr>
<td>2-D Sample Prep for membrane proteins</td>
<td>Reagent</td>
<td>Pierce</td>
<td>89864</td>
<td>&nbsp;</td>
<tr>
<td>Streptomycin sulphate</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

selective labelling of cell-surface proteins using cydye dige fluor minimal dyes

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye  DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan  DIGE technology and analysis of protein expression changes using DeCyder  2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

Watch this Scientific Journal Video about Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes at JoVE.com

Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan&#x2122; DIGE technology.

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of ...

selective-labelling-cell-surface-proteins-using-cydye-dige-fluor

Research

JoVE Journal

Biology

15.1K vues.  GE Healthcare Bio-Sciences AB. Une méthode simple et spécifique a été démontrée pour le marquage fluorescent et une meilleure détection des protéines de surface cellulaire sans une étape de fractionnement. L'abondance différentielle des protéines de surface cellulaire a été analysée par électrophorèse bidimensionnelle (2-D) et Ettan ™ technologie DIGE.

Vidéo: Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons (10 to 25 &mu;m in diameter) are arranged in plexuses that are confined to distinct planes of the gut wall 1; the myenteric plexus can be found between the longitudinal and circular muscle layers, and the submucous plexus between the circular muscle layer and the mucosa. Since the effector systems for these plexuses (transporting epithelium, endocrine cells, immune elements, blood vessels and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve individual components of different reflex pathways. The behavior of the effector systems is controlled by the submucous and myenteric plexuses acting in concert. Therefore, detailed knowledge of synaptic interactions within and between ganglia, and of communication between the plexuses, is essential for understanding normal gastrointestinal function. The ENS, as an intact nervous system, is a unique experimental model in which one can correlate molecular and cellular events with the electrical behavior of the neuronal network and its physiological outputs. Because of the quasi-two-dimensional organization of its plexuses, the ENS is particularly well suited for the study of neural networks using multiple site optical recording techniques that employ voltage-sensitive dyes 2,7,8,9. We will illustrate here the use of a relatively new naphthylstyryl-pyridinium dye (di-4-ANEPPDHQ) 3 that offers multiple advantages over its predecessors, including very low phototoxicity, slow rate of internalization, and remarkable chemical stability. When used in conjunction with a camera that permits sub-millisecond time resolution, this dye allows us to monitor the electrical activity of all the neurons in the field of view with a maximal spatial resolution of ~ 2.5 &mu;m at 100X magnification. At lower magnification (10X or 20X), the sacrifice of single-cell resolution is compensated by a gain in perspective, revealing the intricacies of the inter-ganglionic circuitry.

This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

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 ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...