A Peptide Microarray Technology for Specificity Profiling of Antibodies

Abstract

Source: Cornett, E. M., et al. Analysis of Histone Antibody Specificity with Peptide Microarrays. J. Vis. Exp. (2017).

This video demonstrates a technique that applies peptide microarray technology to profile the specificity of antibodies recognizing post-translational modifications (PTMs) on histone peptides. A microarray slide containing immobilized fluorophore-conjugated histone peptides with known PTM combinations is treated with target PTM-specific antibodies. This process helps identify the antibody's specificity for the target and its potential cross-reactivity with non-target PTMs.

Protocol

1. Designing the Array Slide and Source Plate Layout Under the 'Plan a slide layout' heading on the ArrayNinja interface, click on the link that corresponds to the microarray printer being used. NOTE: ArrayNinja has been programmed to mimic the robotic movement of two commonly used microarray printers (see Table 1). Compatibility with the other arrayers can be configured upon request. Click inside the 'Load plate' dialog …

Representative Results

Figure 1: ArrayNinja Design Module. A screen shot of the ArrayNinja design module is shown in the dotted line. The control panel (top) shows all of the parameters that can be altered on the microarray printer. As these parameters are adjusted, the cartoon image of the slide layout (bottom left) updates in real time. After the layout is set, the user can mouse over individual spots to enter unique…

Materials

Printing Buffer	ArrayIt	PPB
BSA	Omnipure	2390
Streptavidin-coated glass microscope slides	Greiner Bio-one	439003-25
polypropylene 384 well plate	Greiner Bio-one	784201
Biotin-fluorescein	Sigma	53608
contact microarray printer	Aushon	2470	Aushon 2470 Microarray Printer
contact microarray printer	Gene Machines	OmniGrid 100	OmniGrid Microarray Printer
PBS	Invitrogen	14190
Blocking Buffer	ArrayIt	SBB
Hydrophobic wax pen	Vector Labs	H-4000	ImmEdge Hydrophobic Barrier PAP Pen
Silicon Gasket	Grace Bio-labs	622511
Hybridization Vessel	Thermo Scientific	267061	or similar vessel
Fluorescent-dye conjugated secondary antibody	Life Technologies	A-21244	Alexa Fluor 647 (anti-rabbit)
Fluorescent-dye conjugated secondary antibody	Life Technologies	A-21235	Alexa Fluor 647 (anti-mouse)
Wax Imprinter	ArrayIt	MSI48
Tween-20	Omnipure	9490
Microarray Scanner	Innopsys	InnoScan 1100AL	or equivalent microarray scanner
EipTitan Histone Peptide Microarray	Epicypher	112001
AbSurance Pro Histone Peptide Microarray	Millipore	16668
MODified Histone Peptide Array	Active Motif	13001
Histone Code Peptide Microarrays	JPT	His_MA_01
Wax	Royal Oak	GulfWax	for wax imprinter
Humidified Microarray Slide Hybridization Chamber	VWR	97000-284
High throughput microscope slide washing chamber	ArrayIt	HTW
Microscope slide centrifuge	VWR	93000-204
Antibody 1	Abcam	8898
Antibody 2	Millipore	07-473
Biotinylated histone peptide	EpiCypher	12-2001	Example peptide. Similar peptides with various modifications are available from several commercial sources.
ImageMagick			https://www.imagemagick.org/script/index.php
ArrayNinja			https://rothbartlab.vai.org/tools/