Detecting Low‐Affinity Extracellular Protein Interactions Using Protein Microarrays

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Low?affinity extracellular protein interactions are critical for cellular recognition processes, but are not generally detected by methods that can be applied in a high?throughput manner. This unit describes a protein microarray platform that significantly improves the throughput of assays capable of detecting transient extracellular protein interactions. These methodological improvements now permit screening for novel extracellular receptor?ligand interactions on a genome?wide scale. Curr. Protoc. Protein Sci. 72:27.5.1?27.5.15. © 2013 by John Wiley & Sons, Inc.

Keywords: receptor?ligand pairs; extracellular protein interactions; AVEXIS; adhesion receptors; transient/weak interactions; high?throughput screening; microarray

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Introduction
Basic Protocol 1: Preparation of Microarray AVEXIS Assay
Support Protocol 1: High‐Throughput Purification of 6His‐Tagged Proteins
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Preparation of Microarray AVEXIS Assay

Materials

Wash buffer (see recipe )
20 µg each of the bait proteins at 400 ng/µl (see protocol 2 )
Dilution buffer (see recipe )
Anti‐tag primary antibody [e.g., Anti‐Cd4 antibody (OX68); Santa Cruz, cat. no. sc‐53042]
Alkaline phosphatase‐conjugated secondary antibody
Substrate 104 (or other alkaline phosphatase substrate; Sigma‐Aldrich)
ELISA substrate buffer (see recipe )
3 M NaOH
Microarray printing buffer (see recipe )
Nitrocefin (Calbiochen)
Anhydrous dimethyl sulfoxide (DMSO)
1× Phosphate‐buffered saline (PBS; see recipe )
Milli‐Q water
Purified biotinylated HRP
Biotinylated anti‐prey antibody
Pin cleaning solution (Genomic Solutions; see recipe )
Microarray blocking buffer (see recipe )
HRP‐conjugated anti‐flag antibody
Tyramide signal amplification (TSA) kit (Life Technologies, cat. no. T30953) containing:
- Labeled tyramide (Component A)
- Hydrogen peroxide (Component F)
- Amplification buffer (Component E)
N₂ gas, optional

Streptavidin‐coated 96‐well plate (Nunc)
Tissue paper
22°C incubator
Aluminum foil
Plate reader (e.g., PHERAStar Plus; BMG Labtec)
30,000‐MWCO (molecular weight cut off) spin concentrator
Streptavidin‐coated microarray slides (Xantec)
−20°C freezer
Centrifuge
V‐bottom 384‐well plates
Microarray spotting machine (MicroGrid II, Digilab)
FAST frame multi‐slide plate with slide holders (Whatman)
Vortex mixer

Support Protocol 1: High‐Throughput Purification of 6His‐Tagged Proteins

Tissue culture supernatants containing proteins of interest
2 M imidazole, pH 7.4
4 M NaCl
20% ethanol
Deionized water
Running buffer (see recipe )
Elution buffer (see recipe )

96‐well His MultiTrap HP Ni‐NTA plates (GE Healthcare)
Plate docking platform (Sun et al., )
50‐ml plastic syringes

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Figures

Figure 27.5.1 Workflows involved in low‐affinity interaction detection by AVEXIS. Bait and prey proteins are produced as soluble recombinant proteins by transiently transfecting a mammalian cell line. The proteins are purified using an oligo‐His tag using a bespoke purification apparatus (the “Protein Press”) and subsequently printed onto glass slides before screening for interactions.

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Figure 27.5.2 Flowchart showing the salient features of the bait and prey proteins, microarray printing and AVEXIS interaction screening. (A ) Design of the monomeric enzymatically monobiotinylated bait and pentamerized, enzyme‐tagged prey proteins. (B ) Construction of the protein microarrays by printing the monomeric biotinylated bait proteins on streptavidin‐coated slides. (C ) Procedure for screening of the protein microarrays with the prey proteins to detect interactions.

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Figure 27.5.3 An image of a processed microarray slide illustrating typical results. Bait proteins were serially diluted and printed onto a microarray (with the most concentrated solutions at the top) before being probed with an appropriate prey. Positive interactions are detected in a concentration‐dependent manner. The slides also contain prey‐positive controls, landing marks (biotinylated HRP), and negative controls.

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Videos

Literature Cited

Literature Cited
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