Protocols and Tools

Our experimental approach utilizes many protocols and tools. Click on any item from the list below for more details.

Antigen Microarrays (the definitive Protein Microarray protocol, by Brian Kidd)

Antigen Array Production

Probing Antigen Arrays

Data Analysis

Reagents, Supplies, & Equipment

Antigen Array Production

  • Dilute peptides, proteins, protein complexes, or other antigens in PBS or H20 to 0.1 - 0.2 mg/mL (up to 30% DMF or DMSO can be used for peptides and proteins that are insoluble in H20 or PBS).
  • Transfer diluted antigens transferred to 384-well "print plates" (7 µL/well). Antibody or other proteins/peptides prelabeled with Cy-3 and / or Cy-5 (diluted to 0.1 µg/µL) can be added to the print plate to serve as marker spots to orient the arrays post-scanning.
  • Using a robotic arrayer and TeleChem International SMP7B Stealth Micro Spotting Pins (Telechem International, Sunnyvale, CA) to print antigens on Poly-L-lysine coated microarray slides (CEL Associates, Pearland, Texas). For optimal results, increased pin sonication between loads (5 x 7 second sonication cycles), between print plates (5 minutes), and at the end of each print (30 minutes) are recommended.
  • Store arrays at 4°in slide boxes sealed in plastic heat-sealable pouches. Reactivity is generally best within 1 month of printing, but printed antigens can retain reactivity for months.

  • Up to 12,000 features/array
  • 200 mM features
  • 50 pg Ag/feature
  • Probe with 1:150 dilutions sera
  • Use common array equipment
Figure. Antigen array production. The pictured robotic arrayer, developed by Patrick Brown, is used to attach putative peptide and protein autoantigens in ordered arrays on derivatized microscope slides.

Antigen Arrays

Blocking and Wash Buffer: 3% FCS in 1X PBS; 0.05% Tween 20

Blocking and Wash Buffer: 3% FCS in 1X PBS; 0.05% Tween 20

Incubation Buffer: 3% FCS in 1X PBS

1. Prepare slides for blocking.

  • circumscribe arrays on glass slides with hydrophobic marker (PAP pen)
  • use diamond scribe or marker pen to number slides


2. Block slides at 4°C for 4 hours to overnight using a stainless steel slide rack.

3.Incubate arrays with diluted human or animal serum samples.

  • most human and murine sera are used at 1:150 dilutions in incubation buffer
  • place slides in histology chamber, array-side up
  • before the array surface dries, put 300 µL of diluted sera on slide within the region bordered by the hydrophobic marker
  • incubate at 4° C for 1 hr

Note: For sample conservation, arrays can be probed with ~30 µL of diluted serum samples under cover slips.

4. Wash #1

  • quick rinse in wash buffer
    2 x 15 min washes in wash buffer with rotation (~40 rpm)


5.Incubate array with secondary antibody

  • place slides in histology chamber, array-side up
  • put 300 µL of diluted 2° Ab onto each slide
  • (label is photosensitive) wrap histology chamber in aluminum foil
  • incubate at 4° C for 45 minutes


6. Wash #2

  • quick rinse in wash buffer
  • 2 x 30 min washes in wash buffer on rotator (~40 rpm)
  • quick rinse in 1X PBS
  • 2 x 20 min washes in 1X PBS on rotator
  • 2 x 15 sec wash in DDI H2O on rotator


7.Dry slides by centrifugation

  • spin for 8 - 10 min at 25° C
  • put slides in opaque slide box for transport and storage (at room temperature)
  • Arrays should be scanned within several days.


8. Scanning slides

  • use Axon Microarray Scanner and Axon GenePix 5.0 software

Data Analysis


  1. GenePix Pro 5.0 Microarray Analysis Software (Axon Instruments, Inc). Acquires antigen feature intensity data in a form compatible with Microsoft Excel..
  2. ScanAlyze2 (Michael Eisen's Laboratory). Converts images contained in *.TIFF files acquired from scanner into *.BMP files.
  3. Cluster and Treeview (Joe DeRisi's Laboratory). Provides hierarchical cluster analysis algorithms and graphical representation of data. (Eisen et al, PNAS, 95:14863-8, 2001)
  4. Significance Analysis of Microarrays (SAM) and Prediction Analysis of Microarrays (PAM) (xRobert Tibshirani and Gilbert Chu). Identifies statistically significant differences in microarray data sets. SAM identifies array features with statistically significant differences in reactivity between sample groups. PAM performs sample classification from antibody reactivity data to provide a list of significant genes whose expression characterizes each sample group. (Tusher et al, PNAS, 98:5116-21, 2001; Tibshirani et al, PNAS, 99:6567-72, 2002)

General Approach

GenePix Pro 5.0 software (Axon Instruments) is used to extract the net median pixel intensities for individual features from the digital images produced by the GenePix 4000B scanner. Normalized median net digital fluorescence units (DFUs) are generated from the median values from 4-12 identical antigen features on each array. Significance Analysis of Microarrays (SAM) is applied to identify antigens with statistically-significant differences in array reactivity between and Predictive Analysis of Microarrays to identify antigens whose reactivity characterizes defined murine or human clinical sample groups. SAM results can be arranged into relationships using Cluster software. Results are displayed using TreeView software.

Reagents, Supplies, & Equipment

Vendor Product Name Quantity Catalog Number
AP Biotech

CyDye Mono-Reactive Dye 5-Pack  (Cy3)

1 kit # PA 23001
AP Biotech CyDye Mono-Reactive Dye 5-Pack  (Cy5) 1 kit # PA 25001
Axon Instruments, Inc. GenePix 4000B Microarray Scanner    
Axon Instruments, Inc. GenePix Pro5.0 Microarray Analysis  Software    
Beckman MicroPlus Multiwell Plate Carrier set of 2 # 362394
CEL Associates (800-537-9339) Slide Box (Capacity 25 slides) 1 box # SB-25
CEL Associates (800-537-9339) Slide Box (Capacity 100 slides) 1 box # SB-100
TeleChem International Inc. Super Epoxy Slides 25 slides / box # SME
Corning Costar 384-well polyproplylene plates 1 case # 6502
Kapak Corporation SealPAK Packages 9.5" x  16" 20 / package # 506
Kapak Corporation SealPAK Packages 6.5" x 8" 75 / package # 402
Jackson Immunoresearch Goat anti-mouse IgG (H+L) 1.5 mg # 115-005-062
Jackson Immunoresearch Goat anti-human IgG+M Cy3 1.5 mg # 109-165-127
Jackson Immunoresearch Goat anti-human IgG+M Cy5 1.5 mg # 109-175-127
Jackson Immunoresearch Goat anti-rat IgG+M Cy3 1.5 mg # 112-165-068
Jackson Immunoresearch Goat anti-rat IgG+M Cy5 1.5 mg # 112-175-068
Jackson Immunoresearch Donkey anti-rabbit IgG Cy3 0.5 mg # 711-165-152
Jackson Immunoresearch Goat anti-mouse IgG+M Cy3 1.5 mg # 115-165-068
Millipore Micron YM-100 100 filters # 42413
Research Products International (800-323-9814) Kiyota Pap Pen 1 pen 195500
Sigma Goat anti-human IgG 0.25 ml # P-9170
Sigma Goat anti-human IgM 0.25 ml # P-9295
Sigma Human IgM 1 mg # I-8260
Sigma Human IgG 10 mg # I-2511
Sigma Polyoxyethylene Sorbitan Monolaurate (Tween 20) 500 ml # P-1379
TeleChem International Inc. Micro-spotting pins 18 pins # SMP7B
The Binding Site Magnetic Staining (Histology) Chamber 1 chamber # BD010
Thermo Electron Staining dish 3 dishes # 122
Thermo Electron Cover for staining dish, staining dish, 30-slide stainless steel rack 1 assembly # 121
VWR Diamond Scriber 1 scriber # 52865-005
VWR Wire handle for slide rack 1 handle # WLS2499-DB
VWR Dust-off Plus 10 oz can # 21899-094