Reverse Phase Protein Arrays for Compound Profiling

Nathan Moerke1, Mohammad Fallahi‐Sichani2

1 Harvard Medical School—ICCB‐Longwood Screening Facility, Boston, Massachusetts, 2 Harvard Medical School—Department of Systems Biology, Boston, Massachusetts
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.9
Online Posting Date:  September, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Reverse phase protein arrays (RPPAs), also called reverse phase lysate arrays (RPLAs), involve immobilizing cell or tissue lysates, in small spots, onto solid supports which are then probed with primary antibodies specific for proteins or post‐translational modifications of interest. RPPA assays are well suited for large‐scale, high‐throughput measurement of protein and PTM levels in cells and tissues. RPPAs are affordable and highly multiplexable, as a large number of arrays can readily be produced in parallel and then probed separately with distinct primary antibodies. This article describes a procedure for treating cells and preparing cell lysates, as well as a procedure for generating RPPAs using these lysates. A method for probing, imaging, and analyzing RPPAs is also described. These procedures are readily adaptable to a wide range of studies of cell signaling in response to drugs and other perturbations. © 2016 by John Wiley & Sons, Inc.

Keywords: antibodies; BRAF; cell signaling; fluorescence; melanoma; reverse phase protein arrays

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Generation of Lysates for Reverse Phase Protein Arrays
  • Basic Protocol 2: Production of Reverse Phase Protein Arrays
  • Basic Protocol 3: Immunostaining, Imaging, and Analysis of Reverse Phase Protein Arrays
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Generation of Lysates for Reverse Phase Protein Arrays

  Materials
  • Compounds being studied: e.g., PLX‐4032
  • Dimethyl sulfoxide (DMSO)
  • Melanoma cells grown in dishes: e.g., RVH‐421
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.25% trypsin/EDTA (available from many suppliers)
  • Complete medium (see recipe)
  • RPPA lysis buffer (see recipe)
  • Polypropylene 384‐well compound storage plates (Thermo Scientific, cat. no. AB‐1056)
  • Centrifuge
  • Clear 96‐well tissue culture plates
  • Pin transfer apparatus (V&P Scientific, http://www.vp‐scientific.com; Rudnicki and Johnston, )
  • 24‐pin aspiration wand (Drummond)
  • Matrix WellMate (or other automated liquid dispenser for multiwall plates; Rudnicki and Johnson, )
  • WellMate manifold (sterile, optional)
  • Multichannel pipettor
  • Orbital shaker
  • Metal foil seals for microtiter plates (Corning, cat. no. 6570)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise indicated.

Basic Protocol 2: Production of Reverse Phase Protein Arrays

  Materials
  • Lysate plates ( protocol 1)
  • 96‐well filter plates (Pall, cat. no. 5042)
  • 96‐well lysate collection plates (Corning, cat. no. 3870)
  • Multichannel pipettor
  • Centrifuge with microtiter plate carrier
  • Polypropylene 384‐well lysate storage plates (ABgene storage plates; Thermo Scientific, cat. no. AB‐1056)
  • Arraying robot (e.g., Aushon 2470)
  • ONCYTE AVID nitrocellulose coated single‐pad slides (Grace Biolabs, cat. no. 305177)

Basic Protocol 3: Immunostaining, Imaging, and Analysis of Reverse Phase Protein Arrays

  Materials
  • Arrays to be immunostained and imaged (see protocol 2)
  • PBS‐T (see recipe)
  • RPPA wash buffer: 100 mM Tris⋅Cl, pH 9.0
  • Odyssey Blocking Buffer (LI‐COR, cat. no. 927‐40010)
  • Primary antibodies:
    • Rabbit anti‐Erk1/2 phospho‐T204/T205 antibody (Cell Signaling Technologies, cat. no. 4370)
    • Rabbit anti‐rpS6 phospho‐S235/S236 antibody (Cell Signaling Technologies, cat. no. 4848)
    • Mouse anti–β actin antibody (Sigma, cat. no. A1978)
  • Secondary antibodies:
    • Goat anti‐mouse IgG antibody, DyLight 680 conjugated (Pierce, cat. no. 35518)
    • Goat anti‐rabbit IgG antibody, DyLight 800 conjugated (Pierce, cat. no. 35571)
  • Perfect Western 6‐Chamber Incubation Tray (GenHunter, cat. no. B131)
  • Orbital shaker
  • 15‐ and 50‐ml conical tubes (e.g., Corning Falcon)
  • Forceps
  • Centrifuge
  • Near‐infrared‐capable scanner (e.g., InnoScan 710‐IR, Innopsys)
  • Microarray analysis software (e.g., Mapix from Innopsys)
  • Spreadsheet software (e.g., Microsoft Excel)
  • Multidimensional data visualization software (e.g., Matlab and DataPflex)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Albeck, J.G., MacBeath, G., White, F.M., Sorger, P.K., Lauffenburger, D.A., and Gaudet, S. 2006. Collecting and organizing systematic sets of protein data. Nat. Rev. Mol. Cell Biol. 7:803‐812. doi: 10.1038/nrm2042.
  Bollag, G., Tsai, J., Zhang, J., Zhang, C., Ibrahim, P., Nolop, K., and Hirth, P. 2012. Vemurafenib: The first drug approved for BRAF‐mutant cancer. Nat. Rev. Drug. Discov. 11:873‐886. doi: 10.1038/nrd3847.
  Fallahi‐Sichani, M., Moerke, N J., Niepel, M., Zhang, T., Gray, N.S., and Sorger, P.K. 2015. Systematic analysis of BRAF(V600E) melanomas reveals a role for JNK/c‐Jun pathway in adaptive resistance to drug‐induced apoptosis. Mol. Syst. Biol. 11:797. doi: 10.15252/msb.20145877.
  Flaherty, K.T. 2010. Narrative review: BRAF opens the door for therapeutic advances in melanoma. Ann. Intern. Med. 153:587‐591. doi: 10.7326/0003‐4819‐153‐9‐201011020‐00008.
  Gujral, T.S., Karp, R.L., Finski, A., Chan, M., Schwartz, P.E., MacBeath, G., and Sorger, P. 2013. Profiling phospho‐signaling networks in breast cancer using reverse‐phase protein arrays. Oncogene 32:3470‐3476. doi: 10.1038/onc.2012.378.
  Hennessy, B.T., Lu, Y., Gonzalez‐Angulo, A.M., Carey, M.S., Myhre, S., Ju, Z., Davies, M.A., Liu, W., Coombes, K., Meric‐Bernstam, F., Bedrosian, I., McGahren, M., Agarwal, R., Zhang, F., Overgaard, J., Alsner, J., Neve, R.M., Kuo, W.L., Gray, J.W., Borresen‐Dale, A.L., and Mills, G.B. 2010. A technical assessment of the utility of reverse phase protein arrays for the study of the functional proteome in non‐microdissected human breast cancers. Clin. Proteomics 6:129‐151. doi: 10.1007/s12014‐010‐9055‐y.
  Knight, Z.A., Lin, H., and Shokat, K.M. 2010. Targeting the cancer kinome through polypharmacology. Nat. Rev. Cancer 10:130‐137. doi: 10.1038/nrc2787.
  Lee, M.J., Ye, A.S., Gardino, A.K., Heijink, A.M., Sorger, P.K., MacBeath, G., and Yaffe, M.B. 2012. Sequential application of anticancer drugs enhances cell death by rewiring apoptotic signaling networks. Cell 149:780‐794. doi: 10.1016/j.cell.2012.03.031.
  Luckert, K., Gujral, T.S., Chan, M., Sevecka, M., Joos, T.O., Sorger, P.K., Macbeath, G., and Pötz, O. 2012. A dual array‐based approach to assess the abundance and posttranslational modification state of signaling proteins. Sci. Signal 5:pl1. doi: 10.1126/scisignal.2002372.
  Nielsen, U.B., Cardone, M.H., Sinskey, A.J., MacBeath, G., and Sorger, P.K. 2003. Profiling receptor tyrosine kinase activation by using Ab microarrays. Proc. Natl. Acad. Sci. U.S.A. 100:9330‐9335. doi: 10.1073/pnas.1633513100.
  Rudnicki, S. and Johnston, S. 2009. Overview of liquid handling instrumentation for high‐throughput screening applications. Curr. Protoc. Chem. Biol. Dec 1:43‐54. doi: 10.1002/9780470559277.ch090151.
  Sevecka, M. and MacBeath, G. 2006. State‐based discovery: A multidimensional screen for small‐molecule modulators of EGF signaling. Nat. Methods 3:825‐831. doi: 10.1038/nmeth931.
  Sevecka, M., Wolf‐Yadlin, A., and MacBeath, G. 2011. Lysate microarrays enable high‐throughput, quantitative investigations of cellular signaling. Mol. Cell Proteomics. 10:M110 005363. doi: 10.1074/mcp.M110.005363.
  Stiffler, M.A., Grantcharova, V.P., Sevecka, M., and MacBeath, G. 2006. Uncovering quantitative protein interaction networks for mouse PDZ domains using protein microarrays. J. Am. Chem. Soc. 128:5913‐5922. doi: 10.1021/ja060943h.
  Wagner, J.P., Wolf‐Yadlin, A., Sevecka, M., Grenier, J.K., Root, D.E., Lauffenburger, D.A., and MacBeath, G. 2013. Receptor tyrosine kinases fall into distinct classes based on their inferred signaling networks. Sci. Signal 6:ra58. doi: 10.1126/scisignal.2003994.
  Wolf‐Yadlin, A., Sevecka, M., and MacBeath, G. 2009. Dissecting protein function and signaling using protein microarrays. Curr. Opin. Chem. Biol. 13:398‐405. doi: 10.1016/j.cbpa.2009.06.027.
  Zhang, J., Yang, P.L., and Gray, N.S. 2009. Targeting cancer with small molecule kinase inhibitors. Nat. Rev. Cancer 9:28‐39. doi: 10.1038/nrc2559.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library