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
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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

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

  • 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‐; 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

  • 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

  • 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)
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Literature Cited

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