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PROTOCOLS
Proteome Profiler™ antibody arrays adapted for use with the LI-COR® system
Greta Wegner, Ph.D.; Benjamin Cook; Christopher Buehl; and Kathy Brumbaugh, Ph.D.
Sponsored,vendor-submitted protocol    Sponsored by R&D Systems, Inc.    Published in November 2010 (p.29) DOI: 10.2144/000113572

Abstract

Proteome Profiler Antibody Arrays are rapid, sensitive, and economical assays for the simul­taneous detection of multiple secreted and intracellular proteins. Originally developed for chemiluminescence detection, select arrays can now be adapted for the LI-COR Odyssey® Infrared Imaging System. Using this technique, protein expression can be quantified over a much wider linear dynamic range and eliminates the need for film development.

Materials

  • Proteome Profiler Human Cytokine Array (Cat. no. ARY005) (included: Array Buffer 4 and 5, Wash Buffer, Cocktail to each prepared sample. Mix Detection Antibody Cocktail, 4-well Multi-dish).
  • IRDye 800CW Streptavidin (LI-COR, Cat. no. 926-32230)
  • LI-COR Odyssey Infrared Imaging System
  • Sample (serum, plasma, cell culture supernatant)
  • Phosphate-buffered Saline
  • Rocking platform
  • Microcentrifuge

Methods

Reagent preparation

1. Bring all reagents to room temperature before use.

2. Reconstitute the Detection Antibody Cocktail with 100 µL deionized or distilled water.

3. For 1× wash buffer, dilute 40 mL 25× Wash Buffer into 960 mL deionized or distilled water.

Experimental Protocol

4. Using flat-tip tweezers, remove each membrane from between the protective sheets. Carefully cut off the stamped identification number on the membrane using scissors.

5. For blocking, place one membrane in each well of the 4-well Multi-dish containing 2.0 mL Array Buffer 4 and incubate for 1 h on a rocking platform.

6. Prepare samples by adding = 1 mL each sample to 0.5 mL Array Buffer 4. Adjust to a final volume of 1.5 mL with Array Buffer 5 as necessary. Add 15 µL reconstituted Detection Antibody and incubate at room temperature for one hour.

7. Aspirate Array Buffer 4 from thewells of the 4-well Multi-dish and add sample/antibody mixtures prepared in Step 6. Incubate overnight at 2–8°C . on a rocking platform. A shorter incubation time may be used if it is more optimal.

8. Transfer membranes to individual plastic containers with 20 mL 1× Wash Buffer. Rinse the 4-well Multi-dish with deionized or distilled water and dry thoroughly. Wash each membrane with 1× Wash Buffer three times for 10 min on a rocking platform.

9. Dilute the IRDye 800CW Strepta­vidin 1:2000 in Array Buffer 5. Pipet 1.5 mL diluted IRDye 800CW Strepta­vidin into each well of the 4-well Multi-Dish.

10. Transfer each membrane to the 4-well Multi-dish containing the IRDye 800CW Streptavidin and incubate for 30 min at room temperature on a rocking platform.

11. Wash each array as described in Step

8. After washing, drain excess Wash Buffer from the membrane and collect images with an Odyssey Imager.

Data Analysis

Suggested starting LI-COR Odyssey scan parameters:

• Resolution: 84 µm

• Quality: Medium

• Focus offset: 0.0 mm

• Intensity: 5 (adjust as necessary) Collected images may be manipulated using

Adjust Image Curve and Alter Image Display Odyssey settings.

Results

Human peripheral blood mononuclear cells (PBMCs) respond with a significant increase in the production of several cytokines following treatment with the Protein Kinase C activator, phorbol 12-myristate 13-acetate (PMA; Figure 1).

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