This work was funded by R&amp;D Systems, Inc.

1. Sample Preparation
<ol>
	<li>Collect lysates from untreated, ligand treated, or inhibitor and ligand treated cells following the guidelines in the Human Phospho-RTK array datasheet. The lysis buffer has been optimized for this kit. The substitution of other lysis buffers may affect the final performance of the array. To prevent proteolytic sample degradation, add 10 &mu;g/ml Aprotinin, 10 &mu;g/ml Leupeptin, and 10 &mu;g/ml Pepstatin A to the volume of lysis buffer required for each cell lysate preparation fresh for each use.</li>
	<li>Lysate concentration should be measured using a bicinchoninic acid (BCA) assay. Sample concentration may be empirically adjusted for optimal sensitivity and low background. A range of 100-300 &mu;g of lysate is recommended as an initial starting point.</li>
	<li>Freeze/thaw cycles of samples should be avoided and proper storage of lysates is required for optimal performance. Thaw frozen lysate samples on ice.</li>
</ol>
2. Array Assay
<ol>
	<li>Bring all kit components to room temperature before use. To avoid contamination, wear gloves while performing the procedures.</li>
	<li>Pipette 2.0 ml of Array Buffer 1 into each well of the 4-Well Multi-dish to be used. Array Buffer 1 serves as a block buffer.</li>
	<li>Using flat-tip tweezers, remove each membrane to be used from between the protective sheets and place in a well of the 4-Well Multi-dish. The array number should be facing upward.</li>
	<li>Upon contact with Array Buffer 1, the blue dye from the spots will disappear, but the capture antibodies are retained in their specific locations.</li>
	<li>Incubate for 1 hr on a rocking platform. Orient the tray so that each array rocks end to end in its well. The surface of the array should be completely wetted and covered by block buffer.</li>
	<li>While the membranes are blocking, dilute the desired quantity of cellular lysate to a final volume of 1.5 ml with Array Buffer 1.</li>
	<li>Aspirate Array Buffer 1 from the wells of the 4-Well Multi-dish and add diluted lysate solutions. Place the lid on the 4-Well Multi-dish.</li>
	<li>Incubate overnight at 2-8 &deg;C on a rocking platform. A shorter incubation time may be used if optimal sensitivity is not required.</li>
	<li>Carefully remove each membrane from the 4-Well Multi-dish and place into individual plastic containers with 20 ml of 1X Wash Buffer. The 4-Well Multi-dish may not be used to complete wash steps. Rinse the 4-Well Multi-dish with deionized or distilled water and dry thoroughly.</li>
	<li>Wash each membrane with 1X Wash Buffer for 10 min on a rocking platform shaker. Repeat two times for a total of 3 washes.</li>
	<li>Dilute the anti-phospho-tyrosine-horseradish peroxidase (HRP) detection buffer in 1X Array Buffer 2 using the dilution factor on the vial label. Pipette 2.0 ml of diluted solution into each well of the 4-Well Multi-dish.</li>
	<li>Carefully remove each membrane from its wash container. Allow excess buffer to drain from the membrane and return the membrane to the 4-Well Multi-dish containing the diluted anti-phospho-tyrosine-HRP. Cover the wells with the lid.</li>
	<li>Incubate for 2 hr at room temperature on a rocking platform.</li>
	<li>Wash each array as described previously.</li>
	<li>Complete the remaining steps without interruption. Carefully remove each membrane from its wash container. Allow excess buffer to drain from the membrane by blotting the lower edge onto absorbent paper. Place each membrane on a plastic sheet protector with the identification number facing up.</li>
	<li>Pipette 1 ml of the prepared Chemi Reagent Mix evenly onto each membrane. Using less than 1 ml of Chemi Reagent Mix per membrane may result in incomplete membrane coverage. Substitution of some high intensity chemiluminescent reagents for Chemi Reagents 1 and 2 may cause either increased background or diminished signal depending on the reagent.</li>
	<li>Carefully cover with a plastic sheet protector. Gently smooth out any air bubbles and ensure Chemi Reagent Mix is spread evenly to all corners of each membrane. Incubate for 1 min.</li>
	<li>Position paper towels on top and sides of plastic sheet protector containing the membranes and carefully squeeze out excess Chemi Reagent Mix.</li>
	<li>Remove the top plastic sheet protector and carefully lay an absorbent lab wipe on top of the membranes to blot off any remaining Chemi Reagent Mix.</li>
	<li>Leaving membranes on the bottom plastic sheet protector, cover the membranes with plastic wrap taking care to gently smooth out any air bubbles. Wrap the excess plastic wrap around the back of the sheet protector so that the membranes and sheet protector are completely wrapped.</li>
	<li>Place the membranes with the identification numbers facing up, in an autoradiography film cassette that is not used with radioactive isotope detection.</li>
	<li>Expose to Kodak BioMax Light film for 1-10 min. Multiple exposures are recommended. Alternatively, images may be collected using a chemiluminescence compatible imager such as the Carestream Health Image Station 4000MM PRO.</li>
</ol>
3. Data Analysis
<ol>
	<li>Positive signals seen on developed film can be quickly identified by placing the transparency overlay on the array image and aligning it with the pairs of reference spots printed in the corners for this purpose. The stamped identification number on the array should be placed on the left had side. The location of controls and capture antibodies is listed in the Appendix of the Human Phospho-RTK array datasheet.</li>
	<li>Pixel densities on developed film can be collected using a transmission-mode scanner such as the Epson Perfection V750 PRO.</li>
	<li>Create a template to analyze pixel density in each spot of the array using an appropriate imaging analysis software program. Compatible programs include ImageQuant or ArrayVision software from GE, Western Vision software with templates specific to Proteome Profiler Arrays, BioRad Quantity One, Adobe Photoshop, NIH ImageJ, and Protein Simple AlphaView.</li>
	<li>Export signal values to a spreadsheet file for manipulation in a program such as Microsoft Excel.</li>
	<li>Determine the average signal (pixel density) of the pair of duplicate spots representing each RTK.</li>
	<li>Subtract an averaged background signal from each spot. Use a signal from a clear area of the array or negative control spots as a background value.</li>
	<li>Compare corresponding signals on different arrays to determine the relative change in RTK phosphorylation levels between samples.</li>
</ol>
4. Representative Results
This protocol demonstrates how the Human Phospho-RTK array may be used to screen the effects of ligand stimulation and inhibitors on RTK phosphorylation. In Figure 1, data is shown for MDA-MB-453 cells that were untreated, treated with 100 ng/ml rhNRG-&beta;1/HRG-&beta;1 (HRG-&beta;1) for 5 min, or pre-treated with known ErbB family inhibitors8-11 prior to HRG&beta;1 treatment. For inhibitor experiments, the cells were incubated with 1 &mu;M HDS 029, 200 nM PD 153035, or 200 nM PD 158780 in SFM for three hours. Each array was incubated with 100 &mu;g of lysate. An increase in phosphorylation is observed for ErbB2, ErbB3, and ErbB4 capture spots when comparing untreated cells with HRG-&beta;1 treated MDA-MB-453 cells. The treatment of cells with all three ErbB family selective inhibitors prior to HRG-&beta;1 incubation caused reductions in ErbB2, ErbB3, and ErbB4 phosphorylation.
In Figure 2, data is shown for Kato III cells known to overexpress the RTK FGF R2. In this set of experiments, Kato III cells were untreated, treated with 100 ng/ml rhFGF acidic (FGF) and 1 &mu;g/ml heparin, or pretreated with FGF R selective inhibitors12-15 prior to treatment with FGF and heparin. The inhibitors PD173074, PD 161570, and PD 166285 were used at a concentration of 1 &mu;M and incubated with the Kato III cells for 3 hr in serum free media. While there is constitutive phosphorylation of both EGF R and FGF R2 in untreated KATO III cells, an increase in FGF R2 phosphorylation was observed upon FGF treatment. Incubating the cells with PD 173074, PD 161570, or PD 166285 resulted in decreased FGF R2 and EGF R phosphorylation. Although these inhibitors are known affect the phosphorylation of FGF R family members, these results demonstrate the utility of the Human Phospho-RTK array for monitoring the effect a specific concentration of inhibitor may have on other RTKs, such as EGF R in this case.
<img alt="Figure 1" fo:content-width="4.5in" fo:src="/files/ftp_upload/4199/4199fig1highres.jpg" src="/files/ftp_upload/4199/4199fig1.jpg" /> 
 Figure 1. Induction and inhibition of receptor tyrosine kinase phosphorylation in breast cancer cells. Array images from Proteome Profiler Human Phospho-RTK arrays and the corresponding histogram profiles are shown. MDA-MB-453 cells were untreated or treated with RTK inhibitors followed by treatment with NRG-&beta;1/HRG-&beta;1. The array was used to monitor the effects of inhibitors on kinase phosphorylation in MDA-MB-453 cells. <a href="http://www.jove.com/files/ftp_upload/4199/4199fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" fo:content-width="4.5in" fo:src="/files/ftp_upload/4199/4199fig2highres.jpg" src="/files/ftp_upload/4199/4199fig2.jpg" /> 
 Figure 2. Induction and inhibition of receptor tyrosine kinase phosphorylation in gastric cancer cells. Array images from a Proteome Profiler Human Phospho-RTK array and the corresponding histogram profiles are shown. Kato III cells were untreated or treated with RTK inhibitors followed by treatment with FGF acidic and heparin. <a href="http://www.jove.com/files/ftp_upload/4199/4199fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
References
<ol>
 <li>Henriksson, M.L., Edin, S., Dahlin, A.M., Oldenborg, P.A., Oberg, A., Van Guelpen, B., Rutegard, J., Stenling, R., &amp; Palmqvist, R. Colorectal cancer cells activate adjacent fibroblasts resulting in FGF1/FGFR3 signaling and increased invasion. Am. J. Pathol. 178, 1387-1394 (2011).</li>
 <li>Morishita, A., Gong, J., Nomura, T., Yoshida, H., Izuishi, K., Suzuki, Y., Kushida, Y., Haba, R., D'Armiento, J., &amp; Masaki, T. The use of protein array to identify targetable receptor tyrosine kinases for treatment of human colon cancer. Int. J. Oncol. 37, 829-835 (2010).</li>
 <li>Agaram, N.P., Laquaglia, M.P., Ustun, B., Guo, T., Wong, G.C., Socci, N.D., Maki, R.G., DeMatteo, R.P., Besmer, P., &amp; Antonescu, C. R. Molecular characterization of pediatric gastrointestinal stromal tumors. Clin. Cancer Res. 14, 3204-3215 (2008).</li>
 <li>Stommel, J.M., Kimmelman, A.C., Ying, H., Nabioullin, R., Ponugoti, A.H., Wiedemeyer, R., Stegh, A.H., Bradner, J.E., Ligon, K.L., Brennan, C., Chin, L., &amp; DePinho, R.A. Coactivation of receptor tyrosine kinases affects the response of tumor cells to targeted therapies. Science. 318, 287-290 (2007).</li>
 <li>Agarwal, S., Zerillo, C., Kolmakova, J., Christensen, J.G., Harris, L.N., Rimm, D.L., DiGiovanna, M.P., &amp; Stern, D.F. Association of constitutively activated hepatocyte growth factor receptor (Met) with resistance to a dual EGFR/Her2 inhibitor in non-small-cell lung cancer cells. Br. J. Cancer. 100, 941-949 (2009).</li>
 <li>Dewaele, B.M., Floris, G., Finalet-Ferreiro, J., Fletcher, C.D., Coindre, J.M., Guillou, L., Hogendoorn, P.C., Wozniak, A., Vanspauwen, V., Schoffski, P., Marynen, P., Vandenbergh, P., Sciot, R., &amp; Debiec-Rychter, M. Co-activated PDGFRA and EGFR are potential therapeutic targets in intimal sarcoma. Cancer Res. 70, 7304-7314 (2010).</li>
 <li>Yoon, Y.K., Kim, H.P., Han, S.W., Hur, H.S., Oh, D.Y., Im, S.A., Bang, Y.J., &amp; Kim, T.Y. Combination of EGFR and MEK1/2 inhibitor shows synergistic effects by suppressing EGFR/HER3-dependent AKT activation in human gastric cancer cells. Mol. Cancer Ther. 8, 2526-2536 (2009).</li>
 <li>Fry, D.W., Nelson, J.M., Slintak, V., Keller, P.R., Rewcastle, G.W., Denny, W.A., Zhou, H., &amp; Bridges, A.J. Biochemical and antiproliferative properties of 4-[Ar(alk)ylamino]pyridopyrimidines, a new chemical class of potent and specific epidermal growth factor receptor tyrosine kinase inhibitor. Biochem. Pharmacol. 54, 877-887 (1997).</li>
 <li>Bos, M., Mendelsohn, J., Kim, Y.M., Albanell, J., Fry, D.W., &amp; Baselga, J. PD153035, a tyrosine kinase inhibitor, prevents epidermal growth factor receptor activation and inhibits growth of cancer cells in a receptor number-dependent manner. Clin. Cancer Res. 3, 2099-2106 (1997).</li>
 <li>Stoll, S.W., Kansra, S., Peshick, S., Fry, D.W., Leopold, W.R., Wiesen, J.F., Sibilia, M., Zhang, T., Werb, Z., Derynck, R., Wagner, E.F., &amp; Elder, J.T. Differential utilization and localization of ErbB receptor tyrosine kinases in skin compared to normal and malignant keratinocytes. Neoplasia. 3, 339-350 (2001).</li>
 <li>Klutchko, S.R., Zhou, H., Winters, R.T., Tran, T.P., Bridges, A.J., Althau, I.W., Amato, D.M., Elliott, W.L., Meade, M.A., Roberts, B.J., Fry, D.W., Gonzales, A.J., Harvey, P.J., Nelson, J.M, Sherwood, V., Han, H.K., Pace, G., Smaill, J.B., Denny, W.A., &amp; Showalter, H.D. Tyrosine kinase inhibitors. 19. 6-alknyamides of 4-anilinoquinazolines and 4-anilinopyrido[3,4-d]pyrimidines as irreversible inhibitors of the ErbB family of tyrosine kinase receptors. J. Med. Chem. 49, 1475-1485 (2006).</li>
 <li>Kunii, K., Davis, L., Gorenstein, J., Hatch, H., Yashiro, M., Di Bacco, A., Elbi, C., &amp; Lutterbach, B. FGFR2-amplified gastric cancer cell lines require FGFR2 and Erbb3 signaling for growth and survival. Cancer Res.68, 2340-2348 (2008).</li>
 <li>Zhou, W., Hur, W. McDermott, U., Dutt, A., Xian, W., Ficarro, S.B., Zhang, J., Sharma, S.V., Brugge, J., Meyerson, M., Settleman, J. &amp; Gray, N.S. A structure-guided approach to creating covalent FGFR inhibitors. Chem. Biol. 17, 285-295 (2010).</li>
 <li>Batley, B.L., Doherty, A.M., Hamby, J.M., Lu, G.H., Keller, P., Dahring, T.K., Hwang, O., Crickard, K., &amp; Panek R.L. Inhibition of FGF-1 receptor tyrosine kinase activity by PD 161570, a new protein-tyrosine kinase inhibitor. Life Sci. 62 143-150 (1998).</li>
 <li>Panek, R.L, Lu G.H., Klutchko, S.R., Batley, B.L., Darhing, T.K., Hamby, J.M., Hallak, H., Doherty, A.M., &amp; Keiser, J.A. In vitro pharmacological characterization of PD 166285, a new nanomolar potent and broadly active protein tyrosine kinase inhibitor. J. Pharmacol. Exp. Ther. 283, 1433-1444 (1997).</li>
</ol>

The Human Phospho-RTK is an economical and fast alternative to traditional methods such as immunoprecipitation (IP) Western for screening changes in RTK phosphorylation. Using this method, the relative levels of 42 phospho-RTKs were screened simultaneously using a single sample of MDA-MB-453 or Kato III cell lysate. In addition, this array assay required 2.5 hr of hands-on time, making this method far more time-effective than performing multiple IP-Westerns. By employing a chemiluminescence detection method, no specialized equipment beyond what is typically used to collect Western data was required. Arrays are sensitive and may be used to compare changes in phosphorylation caused by both ligand and inhibitor treatment. This method also allows the evaluation of inhibitor selectivity to off-target RTKs. Positive hits may be further evaluated using a quantitative assay such as an ELISA.
To effectively employ arrays to screen for changes in phosphorylation, some key experimental guidelines should be followed. First, experiments should include appropriate control samples. For example, in these experiments the effect of inhibitors on MDA-MB-453 or Kato III lysates were measured on lysate samples prepared on the same day. Only array data that is collected within the same experiment should be analyzed to minimize variations in signal due to differences in sample handling, incubation time, pipet technique, or wash technique. Comparing signal intensities between two analytes on the same membrane is not recommended, since capture antibodies were not selected to have parallel affinities.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td><a href="http://www.rndsystems.com/product_results.aspx?k=ary001" target="_blank">Human Phospho-RTK Array Kit</a></td>
			<td>R&amp;D Sytems</td>
			<td>ARY001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma</td>
			<td>A6279</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Tocris</td>
			<td>1167</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pepstatin A</td>
			<td>Tocris</td>
			<td>1190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MDA-MB-453 cells</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kato III cells</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td><a href="http://www.rndsystems.com/product_results.aspx?k=396-hb" target="_blank">Recombinant Human NRG1-beta 1/HRG1-beta 1 EGF Domain</a></td>
			<td>R&amp;D Systems</td>
			<td>396-HB-050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td><a href="http://www.rndsystems.com/product_results.aspx?k=232-fa" target="_blank">Recombinant Human FGF acidic (aa 16-155)</a></td>
			<td>R&amp;D Systems</td>
			<td>232-FA-025</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H4784</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HDS 029</td>
			<td>Tocris</td>
			<td>2646</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PD 153035</td>
			<td>Tocris</td>
			<td>1037</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PD 158780</td>
			<td>Tocris</td>
			<td>2615</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PD 173074</td>
			<td>Tocris</td>
			<td>3044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PD 161570</td>
			<td>Tocris</td>
			<td>3724</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PD 166285</td>
			<td>Tocris</td>
			<td>3785</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BCA assay</td>
			<td>Pierce</td>
			<td>23225</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate-Buffer Saline (PBS)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipettes and pipette tips</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Deionized or distilled water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Flat-tipped tweezers</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rocking platform shaker</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plastic containers with the capacity to hold 50 ml</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>For washing arrays</td>
		</tr>
		<tr>
			<td>Plastic transparent sheet protector</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plastic wrap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Absorbent lab wipes</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Autoradiography cassette</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Film developer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kodak BioMax Light Film</td>
			<td>Carestream Health</td>
			<td>178 8207</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Film developer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Image Station 4000MM PRO</td>
			<td>Carestream Health</td>
			<td>&nbsp;</td>
			<td>Chemiluminescence imager compatible with collection of western data may be used instead of film and a film developer.</td>
		</tr>
		<tr>
			<td>Epson Perfection Scanner</td>
			<td>Epson</td>
			<td>V750 PRO</td>
			<td>Flatbed scanner with transparency adaptor capable of transmission mode</td>
		</tr>
		<tr>
			<td>Image Analysis Software</td>
			<td>GE,WesternVision, BioRad, Adobe, NIH, Protein Simple, etc</td>
			<td>&nbsp;</td>
			<td>ImageQuant, ArrayVision, Western Vision, Quantity One, Photoshop, ImageJ, AlphaView, or other compatible program</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Computer capable of running image analysis software and Microsoft Excel.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

profiling changes in receptor tyrosine kinase phosphorylation using antibody arrays - advertisement

The dysregulation of receptor tyrosine kinase (RTK) expression and phosphorylation is frequently associated with the development and metastatic spread of cancerous cells.1-3 Significant effort has been focused on developing inhibitors to target specific RTKs and interrupt the aberrant signaling pathways associated with disease states.4-7 The purpose of this study was to measure the effects of a select number of inhibitors on RTK phosphorylation. This study used the Human Phospho-RTK array, which is a membrane based sandwich immunoassay to monitor increases or decreases in phosphorylation for numerous RTKs simultaneously in a single sample. In this assay, both phosphorylated and unphosphorylated RTKs present in a lysate sample are captured by discrete antibodies printed in duplicate across a nitrocellulose membrane the size of a microscope slide. After washing, the arrays are incubated with anti-phospho-tyrosine-horseradish peroxidase (HRP), which sandwiches with phosphorylated RTKs captured on the array. Following a second wash step, the arrays are incubated with chemiluminescent reagents. Signal generated at each array spot is proportional to the amount of phospho-protein bound by each capture antibody. In these experiments, arrays were used to measure increases in phosphorylation following ligand stimulation of either MDA-MB-453 breast cancer or KATO III gastric carcinoma cells, as well as to characterize decreases in phosphorylation associated with the treatment of cells with inhibitors selective towards ErbB or FGF R family members.

Proteome Profiler antibody arrays are a convenient and cost efficient way to screen for changes in receptor tyrosine kinase (RTK) phosphorylation without performing numerous immunoprecipitation (IP) Westerns. The ARY001 Human RTK array allows for the qualitative measurement of multiple RTKs in a single sample using chemiluminescence detection.

Profiling Changes in Receptor Tyrosine Kinase Phosphorylation using Antibody Arrays - ADVERTISEMENT

The dysregulation of receptor tyrosine kinase (RTK) expression and phosphorylation is frequently associated with the development and metastatic spread of ...

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Monitoring Protein Adsorption with Solid-state Nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids 1-6, the unfolding of proteins 7, and binding affinity of different ligands 8. By coupling these nanopores to the resistive-pulse technique 9-12, such measurements can be done under a wide variety of conditions and without the need for labeling 3. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions 13. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

Research

JoVE Journal

Bioengineering

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Immunology and Infection

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

Neuroscience

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Biology

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders

The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence in adulthood 1,2,3. Inactivating mutations in KISS1R identified in patients have been associated with iodiopathic hypogonadotropic hypogonadism (IHH) 1,2 and precocious puberty 4. Functional studies of these mutants are crucial for our understanding of the mechanisms underlying the regulation of reproduction by this receptor as well as those shaping the disease outcomes, which result from abnormal KISS1R signaling and function. However, the highly GC-rich sequence of the KISS1R gene makes it rather difficult to introduce mutations or amplify the gene encoding this receptor by PCR.
Here we describe a method to introduce mutations of interest into this highly GC-rich sequence that has been used successfully to generate over a dozen KISS1R mutants in our laboratory. We have optimized the PCR conditions to facilitate the amplification of a range of KISS1R mutants that include substitutions, deletions or insertions in the KISS1R sequence. The addition of a PCR enhancer solution, as well as of a small percentage of DMSO were especially helpful to improve amplification. This optimized procedure may be useful for other GC-rich templates as well.
The expression vector encoding the KISS1R is been used to characterize signaling and function of this receptor in order to understand how mutations may change KISS1R function and lead to the associated reproductive phenotypes. Accordingly, potential applications of KISS1R mutants generated by site-directed mutagenesis can be illustrated by many studies 1,4,5,6,7,8. As an example, the gain-of-function mutation in the KISS1R (Arg386Pro), which is associated with precocious puberty, has been shown to prolong responsiveness of the receptor to ligand stimulation 4 as well as to alter the rate of degradation of KISS1R 9. Interestingly, our studies indicate that KISS1R is degraded by the proteasome, as opposed to the classic lysosomal degradation described for most G protein-coupled receptors 9. In the example presented here, degradation of the KISS1R is investigated in Human Embryonic Kidney Cells (HEK-293) transiently expressing Myc-tagged KISS1R (MycKISS1R) and treated with proteasome or lysosome inhibitors. Cell lysates are immunoprecipitated using an agarose-conjugated anti-myc antibody followed by western blot analysis. Detection and quantification of MycKISS1R on blots is performed using the LI-COR Odyssey Infrared System. This approach may be useful in the study of the degradation of other proteins of interest as well.

Mutations in the kisspeptin receptor (KISS1R) are associated with reproductive disorders in patients. Here we describe how to introduce mutations of interest in the GC-rich sequence of KISS1R as well as the use of KISS1R constructs to characterize the degradation pathway of the receptor by immunoprecipitation and western blot.

The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence in ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...