Preparation and Use of Reverse Protein Microarrays

Elisa Pin1, Giulia Federici2, Emanuel F. Petricoin3

1 Division of Experimental Oncology 2, Centro di Riferimento Oncologico‐IRCCS, National Cancer Institute, Aviano, 2 Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità (ISS), Rome, 3 Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, Virginia
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 27.7
DOI:  10.1002/0471140864.ps2707s75
Online Posting Date:  February, 2014
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Abstract

Reverse‐phase protein array (RPPA) is a multiplex, high‐throughput proteomic technique for profiling the activation status of signal transduction pathways involved in cancer survival and progression, potentially allowing for identification of new biomarkers and drug targets. On RPPA, the entire patient proteome is immobilized on a spot and single proteins can be quantified across a set of samples, spotted on the same array, with high specificity and sensitivity. Array immunostaining and signal amplification systems are used to generate a signal proportional to the concentration of the analyte. Dedicated scanners and software are used to detect spots, measure intensity, subtract background, normalize signal, and generate a numeric value as output. The generated output file is then analyzed using several different bioinformatic and biostatistical tools. In this unit, the RPPA procedure is described in depth, from sample handling and preparation to data analysis, with particular emphasis on tissue sample analysis. Curr. Protoc. Protein Sci. 75:27.7.1‐27.7.29. © 2014 by John Wiley & Sons, Inc.

Keywords: tissues; cells; protein; microarray; antibody; analysis

     
 
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Table of Contents

  • Introduction
  • Protein Sample Preparation for Reverse‐Phase Protein Microarray
  • Basic Protocol 1: Lysing Cells in Suspension
  • Basic Protocol 2: Lysing Adherent Cells
  • Tissues
  • Basic Protocol 3: Whole‐Tissue Chunk Lysis
  • Basic Protocol 4: Whole or Laser‐Capture Microdissected Tissue Section Lysis
  • Basic Protocol 5: Extraction of Proteins from Biological Fluids and Low‐Molecular‐Weight Serum Fraction Samples
  • Basic Protocol 6: Total Protein Determination and Preparation for RPPA
  • Basic Protocol 7: Reverse‐Phase Protein Microarray Printing
  • Basic Protocol 8: Microarray Total Protein Determination Using Sypro Ruby
  • Basic Protocol 9: Pre‐Treatment of RPPA Slides for Immunostaining
  • Basic Protocol 10: Microarray Immunostaining: Dako‐Catalyzed Signal Amplification System (CSA)
  • Basic Protocol 11: Scanning Immunostained RPPA Slides and Data Analysis
  • Support Protocol 1: Tissue Section Staining for Whole and LCM Tissue Lysis
  • Support Protocol 2: Cleaning and Priming Aushon Arrayer Pins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Lysing Cells in Suspension

  Materials
  • Fresh cell cultures
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS), ice cold
  • Cell lysis buffer (see recipe), prepared fresh and store on ice
  • 15‐ml and 1.5‐ml collection tubes
  • Refrigerated centrifuge
  • Vortexer

Basic Protocol 2: Lysing Adherent Cells

  Materials
  • Fresh cell cultures
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS), ice cold
  • Cell lysis buffer (see recipe), prepare fresh
  • Flask scrapers
  • 15‐ml and 1.5‐ml collection tubes
  • Refrigerated centrifuge

Basic Protocol 3: Whole‐Tissue Chunk Lysis

  Materials
  • Frozen, whole‐tissue chunks
  • Dry ice
  • Extraction buffer (see recipe), prepare fresh
  • Pestles and appropriate 1.5‐ml vials
  • 100°C dry heat block

Basic Protocol 4: Whole or Laser‐Capture Microdissected Tissue Section Lysis

  Materials
  • Frozen, whole‐tissue sections (8‐µm thick) on glass slides
  • Dry ice
  • Hematoxylin and eosin (H&E) staining solution (see protocol 12)
  • Extraction buffer (see recipe)
  • LCM station for tissue dissection
  • 0.5‐ml screw‐cap tubes
  • 100°C dry heat block

Basic Protocol 5: Extraction of Proteins from Biological Fluids and Low‐Molecular‐Weight Serum Fraction Samples

  Materials
  • LMW protein
  • 20% glycerol
  • 2‐mercaptoethanol (Sigma Aldrich, cat. no. M‐6250; 2.5% final concentration)
  • 2× Novex Tris‐glycine SDS sample buffer (Invitrogen, cat. no. LC2676)
  • LMW protein sample buffer (see recipe)
  • MiniPrep cell apparatus (BioRad, cat. no. 170‐2908)
  • Microcon Ultracel YM‐3 centrifugal filter device (Millipore, cat. no. MRCPRT010)
  • Centrifuge
  • 100°C dry heat block
  • Additional reagents and equipment for SDS‐PAGE and silver nitrate staining

Basic Protocol 6: Total Protein Determination and Preparation for RPPA

  Materials
  • Samples lysed in T‐PER‐based lysis buffer
  • 2× Novex Tris‐glycine SDS sample buffer (Invitrogen, cat. no. LC2676)
  • 5% 2‐mercaptoethanol (2‐ME) (Sigma Aldrich, cat. no. M‐6250) (2.5% final concentration)
  • Samples (to be lysed in extraction buffer)
  • Extraction buffer (see recipe)
  • 2 mg/ml BSA solutions (e.g., Thermo Scientific, cat. no. 23209)
  • Nitrocellulose‐coated glass slide (e.g., Grace BioLabs, Whatman, Scott)
  • Sypro Ruby protein blot stain (Invitrogen, cat. no. S‐11791)
  • 0.5‐ml screw‐cap tubes
  • 100°C dry heat block
  • Vortex
  • Microcentrifuge
  • 384‐well plates
  • Aushon 2470 arrayer (see protocol 7)
  • Microarray analysis software (e.g., Microvigene)
  • GraphPad software

Basic Protocol 7: Reverse‐Phase Protein Microarray Printing

  Materials
  • Extraction buffer (see recipe)
  • Samples and control samples, such as HeLa+Pervanadate cell lysate (BD, cat. no. 612014), Jurkat+Calyculin A cell lysate (Cell Signaling, cat. no. 9273), and Jurkat+Etoposide cell lysate (Cell Signaling, cat. no. 2043)
  • Nitrocellulose‐coated slides (Whatman FAST slides, Schott Nexterion slides small pore size or Grace ONCYTE Avid slides)
  • Drierite crystals
  • Aushon 2470 arrayer
  • 384‐well microtiter plates with lids (Genetix, cat. no. X6003)
  • Membrane marking pen (Fisher, cat. no. NC9520952)
  • Slide box
  • Plastic zipper bags

Basic Protocol 8: Microarray Total Protein Determination Using Sypro Ruby

  Materials
  • Selected RPPA slides
  • Fixative solution (see recipe)
  • Sypro Ruby protein blot stain (Invitrogen, cat. no. S‐11791)
  • Plastic dishes
  • Rocking platform shaker (e.g., Belly Dancer platform shaking, Stoval, cat. no. 53246‐1) or orbital shaker
  • Aluminum foil
  • Laser scanner or another system able to capture fluorescent signals with a Sypro Ruby filter
  • Microarray analyzer software (e.g., MicroVigene, VigeneTech)

Basic Protocol 9: Pre‐Treatment of RPPA Slides for Immunostaining

  Materials
  • I‐Block solution (see recipe)
  • Re‐Blot mild antibody stripping solution (10×) (Chemicon, cat. no. 2502)
  • Selected printed RPPA slides
  • 1× PBS without calcium and magnesium (see recipe)
  • Plastic dishes
  • Rocking platform (e.g., Belly Dancer platform, Stoval, cat. no. 53246‐1) or orbital shaker

Basic Protocol 10: Microarray Immunostaining: Dako‐Catalyzed Signal Amplification System (CSA)

  Materials
  • Selected and validated primary antibodies (see Critical Parameters)
  • Biotinylated secondary antibody:
    • Anti‐mouse (supplied in CSA kit)
    • Anti‐rabbit (Vector, cat. no. BA‐1000)
  • CSA kit (Dako, cat. no. K1500) containing:
    • 3% hydrogen peroxide (ready‐to‐use solution)
    • Protein block solution (ready‐to‐use solution)
    • Anti‐mouse‐linked secondary antibody
    • Streptavidin‐biotin complex
    • Amplification reagent (ready‐to‐use solution)
    • Streptavidine peroxidase (streptavidin‐HRP; ready‐to‐use solution)
  • Antibody diluent (Dako, cat. no. S3022)
  • DAB solution (Dako, cat. no. K3468) for chromogenic method or IRDye680‐streptavidin (LiCor, cat. no. 926‐32231) for fluorescent method
  • 1× PBS without calcium and magnesium (Invitrogen, cat. no. 14190 or see recipe)
  • Bovine serum albumin (BSA powder, Sigma Aldrich, cat. no. A7888‐50G)
  • 10× Tris‐buffered saline with Tween 20 (TBST, Dako, cat. no. S3306 or see recipe)
  • Pre‐treated slides (see protocol 9)
  • Biotin blocking system (consisting of a vial of ready‐to‐use biotin and a vial of ready‐to‐use avidin; Dako, cat. no. X0590)
  • Dako autostainer
  • Autostainer tubes
  • Racks for autostainer tubes

Basic Protocol 11: Scanning Immunostained RPPA Slides and Data Analysis

  Materials
  • RPPA slides
  • Document scanner or fluorescence scanner equipped with emission/excitation filters compatible with fluorophore(s)
  • Image analysis software (e.g., MicroVigene, VigeneTech)

Support Protocol 1: Tissue Section Staining for Whole and LCM Tissue Lysis

  Materials
  • Frozen, 8‐µm thick, whole‐tissue sections on glass slides
  • 70% ethanol containing protease inhibitors
  • dH 2O containing protease inhibitors
  • Hematoxylin containing protease inhibitors
  • Scott's Tap Water containing protease inhibitors
  • 95% and 100% ethanol
  • Xylene
NOTE: Protease inhibitor tablets (e.g., Complete Protease Inhibitor, Roche) are water soluble. Dissolve them in water by vortexing tubes before adding the alcoholic component.

Support Protocol 2: Cleaning and Priming Aushon Arrayer Pins

  Materials
  • BME extraction buffer (see recipe)
  • 384‐well plates
  • Nitrocellulose slides
  • Aushon arrayer
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Figures

Videos

Literature Cited

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