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

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Abstract
Table of Contents
Materials
Figures
Videos
Literature Cited

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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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) H₂ O (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

Figure 27.2.1 Schematic illustrating array features printed with plasmid DNA in the presence of capture antibodies and conversion of cDNA to protein by in vitro transcription and translation. Capture antibodies tether and immobilize proteins by binding to the protein‐fused affinity tag.

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Figure 27.2.2 A representative high‐density slide printed with plasmid DNA converted to protein by treatment with a cell‐free expression system (left). Plot of the DNA signal obtained from Pico Green staining against the protein signal obtained by staining with anti‐GST antibodies illustrates the relatively tight range of both DNA and protein captured on Nucleic Acid Programmable Protein Array (NAPPA). Figure originally published in (Ramachandran, ) and used here with permission.

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Figure 27.2.3 A pro‐plate mini‐array used to titer serum to determine the best dilution for the full‐scale array. The outlined boxes indicate the dilution chose for each serum. The false color scheme is white saturated>red>orange>yellow>green>blue.

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Figure 27.2.4 (A ) Scraped features that would give erroneously low signal. (B ) Regional biases in slide signal intensity most likely due to technical errors in development. (C ) A microscopic fiber on the array surface that spans a single feature would contribute positive error to signal intensity. (D ) Poor spot morphology of features on the left (versus good features on the right) that may arise from drying during serum challenge and development.

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Figure 27.2.5 Plots of the median intensity for all features in a sub‐array or block (printed by a single pin) reveal differences in immunoreactive signal that persist over the majority of the serum‐challenged array (i.e., pin 20, pin 46, pin 35), suggesting pin‐dependent artifacts that may require correction.

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Figure 27.2.6 Representative histogram of log₂ ‐transformed signal intensity from a serum‐challenged NAPPA slide developed with anti‐human secondary antibodies and tyramide signal amplification.

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Figure 27.2.7 Pseudo‐colored images (left) and scatter plots (right) for replicate arrays challenged with the same serum samples on separate days with computed correlation coefficient.

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Figure 27.2.8 Replicate images of two Pico Green‐stained slides from a single print batch (left). Signal‐intensity data extracted from each and plotted against one another show high concordance between values and verify reproducible printing (right).

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Figure 27.2.9 Replicate images of two anti‐GST stained slides from a single print batch developed by amplified labeling (left). Signal‐intensity data extracted from each and plotted against one another show high concordance between values and verify reproducible protein expression and display (right).

View Image

Videos

DNA isolation and array sample preparation

Duration: 9:38

Play Video
Preparation of Master Mix and the arrayer

Duration: 4:21

Play Video
Slide expression, detection, and imaging

Duration: 6:57

Play Video
Aminosilane slide coating

Duration: 2:32

Play Video
Detection of array-immobilized DNA

Duration: 2:18

Play Video

Literature Cited

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

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited