Analysis of Histone Antibody Specificity with Peptide Microarrays

Summary

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

Abstract

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

Introduction

Genomic DNA is elegantly packaged inside the eukaryotic cell nucleus with histone proteins to form chromatin. The repeating subunit of chromatin is the nucleosome, which consists of 147 base pairs of DNA wrapped around an octameric core of histone proteins – H2A, H2B, H3, and H4¹. Chromatin is broadly organized into loosely packed euchromatin and tightly packed heterochromatin domains. The degree of chromatin compaction regulates the extent to which protein machineries can access the underlying DNA to carry out fundamental DNA-templated processes like replication, transcription, and repair.

Key regulators of genome a....

Protocol

1. Installing and Running ArrayNinja

1. Download and install Oracle Virtual Box from www.virtualbox.org.
2. Download and uncompress the ArrayNinja virtual machine (VM) from http://research.vai.org/Tools/arrayninja.
3. Open Virtual Box and add the ArrayNinja VM by clicking 'Machine,' 'Add,' and select arrayninja.vbox from the folder where ArrayNinja VM was saved.
4. Start ArrayNinja by selecting it inside Virtual Box and clicking the green 'start' arrow.
5. Virtual Box will open a new window and display a message that the VM can be accessed by navigating the web browser to localhost:2080.                                             ....

Results

This protocol has been used to design and fabricate a peptide microarray platform for the analysis of histone PTM antibody specificity. The array queries a library of more than 300 unique peptide features (20 - 40 residues in length) representing many of the known combinations of PTMs found on core and variant histone proteins³⁸. This pipeline has been a workhorse for the screening of many widely used and commercially available histone PTM antibodies, and full data.......

Discussion

Antibody reliability in biomedical research applications is paramount^46,47. This is especially true in chromatin biochemistry given the position of antibodies as key tools for the majority of techniques developed to characterize the abundance and distribution of histone PTMs. The protocol presented here details an optimized pipeline for the design, fabrication, and use of peptide microarrays to analyze histone PTM antibody specificity. This pipeline has been used.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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	12-0001	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/