A Peptide Microarray Technology for Specificity Profiling of Antibodies

Published: April 30, 2024

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…

Offenlegungen

The authors have nothing to disclose.

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/

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A Peptide Microarray Technology for Specificity Profiling of Antibodies. J. Vis. Exp. (Pending Publication), e22115, doi: (2024).

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