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

• 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.

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

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)