Nucleic Acid Programmable Protein Arrays: Versatile Tools for Array‐Based Functional Protein Studies

Shane Miersch1, Joshua LaBaer1

1 Biodesign Institute at Arizona State University, Tempe, Arizona
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 27.2
DOI:  10.1002/0471140864.ps2702s64
Online Posting Date:  April, 2011
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Abstract

Protein microarrays offer a global perspective on the function of expressed gene products. However, technical issues related to the stability and dynamic range of microarrays printed with purified protein have hampered their widespread adoption. Taking an alternate approach, the Nucleic Acid Programmable Protein Array (NAPPA) is constructed by spotting protein‐encoding plasmid DNA at high density, in addressable fashion, on an array surface. Proteins are subsequently generated in situ just prior to experimentation using cell‐free expression systems. As such, the NAPPA platform offers a unique and viable alternative that circumvents many of the inherent limitations of spotted protein arrays, enabling diverse functional protein studies including protein–small molecule, protein‐protein, antigen‐antibody, and protein–nucleic acid interactions. It further offers a versatile and adaptable platform amenable to a variety of capture modalities and expression systems, and, most importantly, construction of the array is accessible to any lab with an array printer and laser slide scanner. This unit is intended to provide a reference for investigators wishing to generate arrays based on this platform, and details (1) the basic construction of cDNA‐based protein microarrays from DNA isolation to printing and development, (2) quality‐control efforts taken to ensure the usefulness and integrity of microarray data, and (3) a particular example of the application of self‐assembling protein arrays to screen for blood‐borne antibody biomarkers. Curr. Protoc. Protein Sci. 64:27.2.1‐27.2.26. © 2011 by John Wiley & Sons, Inc.

Keywords: protein microarray; antibodies; serum profiling

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Generation, Isolation and Quantitation of Plasmid DNA for Printing
  • Basic Protocol 2: DNA Precipitation and Preparation of Print Mix for Arraying
  • Basic Protocol 3: On‐Slide Expression, Serum Challenge, and Development
  • Alternate Protocol 1: Direct Labeling
  • Alternate Protocol 2: Amplified Labeling
  • Basic Protocol 4: Image Scanning and Data Collection
  • Support Protocol 1: Slide Coating for Nucleic Acid Programmable Protein Arrays
  • Support Protocol 2: Quantification of DNA Printed on NAPPA Slides
  • Support Protocol 3: Detection of Displayed Protein on NAPPA Slides with Anti‐GST Antibody
  • Support Protocol 4: Pretitration for Signal Stabilization
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generation, Isolation and Quantitation of Plasmid DNA for Printing

  Materials
  • LB agar in Omni plates (see recipe) with appropriate selection antibiotic
  • Recombinant bacterial library transformed with plasmid‐encoded proteins fused to affinity tag (T7 promoter‐based) (e.g., from the Center for Personalized Diagnostics of the Biodesign Institute at Arizona State University; 96‐well plates of bacterial glycerol stock)
  • LB liquid medium (LB broth; BD Difco, cat. no. 95026‐856) or Terrific broth (see recipe), with appropriate selection antibiotic
  • Solution 1: resuspension buffer (see recipe)
  • Solution 2: lysis buffer (see recipe)
  • Solution 3: neutralization buffer (see recipe)
  • NucleoBond anionic resin (Machery Nagel, cat. no. 740503.1, http://www.mn‐net.com/)
  • Solution N2: equilibration buffer (see recipe)
  • Solution N3: wash buffer (see recipe)
  • Solution N5: elution buffer (see recipe)
  • 10× TE buffer: 100 mM Tris⋅Cl, pH 7.5 ( appendix 2E)/10 mM EDTA
  • bisBenzimide H (Hoechst dye; Sigma)
  • Plasmid DNA to prepare DNA standards
  • 96‐pin replicator device (Boekel, cat. no. 140500, http://www.boekelsci.com/)
  • 96‐well deep‐well blocks (Riplates), 2 ml/well, for bacterial cultivation (RK Manufacturing, cat. no. RRK‐850356B, http://www.rkmfg.com/)
  • Gas permeable plate seals (Axygen, cat. no. BF‐400, http://www.axygen.com)
  • ATR Multitron shaker (ATRBiotech, http://www.atrbiotech.com/)
  • Centrifuge with capacity to provide slow ramp of acceleration and to handle 96‐well deep‐well plates
  • Multichannel pipettors or automated multiwell dispenser (e.g., Wellmate, Thermo Scientific)
  • Aluminum plate seal (Axygen, cat. no. PCR‐AS‐200, http://www.axygen.com/)
  • Unifilter 96‐well microplate filter device, 800 µl/well (Whatman, cat. no. 7700‐2804)
  • Vacuum manifold fitted for microwell plate with liquid waste trap (Pall, cat. no. 5017)
  • 96‐well storage (collection) plate, 800 µl/well (ThermoScientific, cat. no. AB0859)
  • Black fluorescent microwell plates (ISC BioExpress, cat. no. T3025‐16, http://www.bioexpress.com/)
  • Fluorescent microwell plate reader equipped with 365/405 nm excitation/emission (Spectramax M5 microplate reader; Molecular Devices)
NOTE:Video 1 illustrates protocol 1 and steps 1 to 4 of protocol 2 (see Video 1 at http://www.currentprotocols.com/protocol/ps2702).

Basic Protocol 2: DNA Precipitation and Preparation of Print Mix for Arraying

  Materials
  • 96‐well plate containing DNA samples ( protocol 1)
  • 3 M potassium acetate, pH 3.5 (e.g., Sigma)
  • Isopropanol (e.g., Sigma)
  • 80% (v/v) ethanol (prepare from 200 proof ethanol, e.g., Sigma)
  • 66 mg/ml bovine serum albumin (BSA; e.g., Sigma)
  • 50 mg/ml bis‐sulfosuccinimdylsuberate (BS3;Pierce, cat. no. PI 21580) in dimethylsulfoxide (DMSO)
  • 5 mg/ml polyclonal anti‐GST capture antibody (Amersham, cat. no. 27‐4577‐01)
  • Aluminum foil plate seal (Axygen, cat. no. PCR‐AS‐200, http://www.axygen.com)
  • Centrifuge with capacity to handle 96‐well deep‐well plates
  • Genetix Q Array 2 robotic microarray printing unit with 300‐µm tungsten contact pins (http://www.genetix.com/)
  • Coated slides ( protocol 7)
  • Genetix 7020 384‐well plates (http://www.genetix.com/)
  • Multichannel pipettors or automated multiwell dispenser (e.g., Wellmate, Thermo Scientific)
  • Eppendorf Thermomixer plate shaker (Fisher Scientific)
NOTE:Video 1 illustrates protocol 1 and steps 1 to 4 of this protocol. Video 2 illustrates steps 5 to 22 of protocol 2. See Videos 1 and 2 at http://www.currentprotocols.com/protocol/ps2702.

Basic Protocol 3: On‐Slide Expression, Serum Challenge, and Development

  Materials
  • Printed slides ( protocol 2)
  • SuperBlock blocking buffer (ThermoScientific, cat. no. 37535)
  • TnT Reticulocyte Lysate In vitro Transcription and Translation system (Promega, cat. no. L4610) including:
    • Amino acid mixture minus Leu
    • Amino acid mixture minus Cys
    • Amino acid mixture minus Met
    • TnT buffer
    • T7 RNA polymerase
    • TnT rabbit reticulocyte lysate
  • DEPC‐treated (nuclease‐free) H 2O (Promega, cat. no. P1193)
  • 5% skim milk in PBST, pH 7.4 (see recipe)
  • Serum to be assayed for antibodies to antigens on the arrays (optional)
  • Hybriwell gaskets with adhesive inlet covers (Grace Bio‐Labs, 44904)
  • Echotherm Chilling Incubator (Sigma‐Aldrich, cat. no. Z400963)
  • Incubation dishes (rectangular 4‐well dishes, Nalgene Nunc, cat. no. 267061)
  • Rocking platform shaker (e.g., VWR. cat. no. 40000‐304)
  • Hybridization chamber (Corning, product no. 2551)
  • Rotating slide rotisserie (Labquake 415110, Thermolyne)
  • Additional reagents and equipment for direct labeling ( protocol 4) or amplified labeling ( protocol 5)
NOTE:Video 3 illustrates steps 1 to 11 of this protocol (see Video 3 at http://www.currentprotocols.com/protocol/ps2702).

Alternate Protocol 1: Direct Labeling

  • Washed slide containing newly expressed proteins (step 14 of protocol 3)
  • Secondary antibody (dilute 1:500): Cy3‐labeled anti‐mouse (Jackson ImmunoResearch, cat. no. 115‐165‐071) or Cy3‐labeled anti‐human (Jackson ImmunoResearch, cat. no. 109‐165‐008)
  • PBST, pH 7.4 (see recipe)
  • Hybridization chamber (Corning, product no. 2551)
NOTE: Procedures in this protocol are illustrated in Video 3 (see Video 3 at http://www.currentprotocols.com/protocol/ps2702).

Alternate Protocol 2: Amplified Labeling

  • Washed slide containing newly expressed protein (step 14 of protocol 3)
  • Secondary antibody (dilute 1:500): HRP‐conjugated anti‐mouse antibody (Jackson ImmunoResearch, cat. no. 115‐035‐071) or HRP‐conjugated anti‐human antibody (Jackson ImmunoResearch, cat. no. 109‐035‐098)
  • TSA reagent and diluent (PerkinElmer, cat. no. SAT704B)
  • Hybridization chamber (Corning, cat. no. 2551)
  • Slide dishes
  • Kimwipes, Delicate Task Wipers
  • Lifter slips, 24× 65 mm (ThermoScientific, cat. no. 25X65I‐2‐5251)
NOTE: Procedures in this protocol are illustrated in Video 3 (see Video 3 at http://www.currentprotocols.com/protocol/ps2702).

Basic Protocol 4: Image Scanning and Data Collection

  Materials
  • 3‐aminopropyltriethoxysilane (ThermoScientific)
  • Acetone (e.g., Sigma)
  • Silica desiccant packets
  • 1 × 2–in. glass slides
  • Metal slide rack
  • Glass coating chambers with lid (of sufficient size to hold a metal rack containing slides)
  • Rocking platform (e.g., VWR, cat. no. 40000‐304)
NOTE: Procedures in this protocol are illustrated in Video 4 (see Video 4 at http://www.currentprotocols.com/protocol/ps2702).

Support Protocol 1: Slide Coating for Nucleic Acid Programmable Protein Arrays

  Materials
  • Slide containing DNA array ( protocol 2)
  • SuperBlock blocking buffer (ThermoScientific, cat. no. 37535)
  • Quanti‐iT Pico Green (Invitrogen)
  • TE buffer, pH 7.5 ( appendix 2E)
  • PBST, pH 7.4 (see recipe)
  • DMSO, Sigma, St. Louis, MO
  • Rocking platform (VWR, cat. no. 40000‐304)
  • Kimwipes, Delicate Task Wipers
  • Incubation dishes (rectangular 4‐well dishes; Nalgene Nunc, cat. no. 267061)
  • Lifter slips, 24× 65 mm (ThermoScientific, cat. no. 25X65I‐2‐5251)
  • Scanner (Tecan Power Scanner, http://www.tecan.com; or equivalent scanner for standard glass slides)
  • Additional reagents and equipment for scanning slides ( protocol 6)
NOTE: Procedures in this protocol are illustrated in Video 5 (see Video 5 at http://www.currentprotocols.com/protocol/ps2702).

Support Protocol 2: Quantification of DNA Printed on NAPPA Slides

  Materials
  • Array slide bearing newly expressed proteins (step 14 of protocol 3)
  • Primary antibody: mouse anti‐GST (dilute 1:500)
  • 5% skim milk in PBST, pH 7.4 (see recipe)
  • Anti‐affinity tag antibodies for development and determination of protein display, 2624B (Cell Signaling Technology)
  • Hybridization chamber (Corning, product no. 2551)
  • Incubation dishes (rectangular 4‐well dishes; Nalgene Nunc, cat no. 267061)
  • Rotating slide rotisserie (Labquake 415110, Thermolyne)
  • Additional reagents and equipment for detection ( protocol 4 or protocol 52)
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Figures

Literature Cited

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