H2AFY2 Antibody

What is H2AFY2 and why is it significant in epigenetic research?

H2AFY2 (also known as macroH2A2, mH2A2) is a variant of the histone H2A family with a distinct structural feature that includes a large macro domain. It replaces conventional H2A in a subset of nucleosomes where it functions as a transcriptional repressor. Its significance in epigenetic research stems from its role in:

• Chromatin organization and compaction

• Stable X chromosome inactivation

• Regulation of transcriptional programs

• Brain development

H2AFY2 contributes to nucleosomes that wrap and compact DNA into chromatin, limiting DNA accessibility to cellular machineries that require DNA as a template . This makes it a critical protein for studying epigenetic regulation mechanisms.

What are the distinguishing characteristics of H2AFY2 compared to other histone variants?

H2AFY2 differs from canonical H2A and other variants in several key aspects:

The most distinctive feature is the presence of the macro domain, which is involved in protein-protein interactions and recognition of ADP-ribose . This feature allows H2AFY2 to participate in specialized chromatin functions beyond the structural role of canonical histones .

How is H2AFY2 expression regulated in different tissue types?

H2AFY2 shows tissue-specific expression patterns, contrary to canonical histones which are ubiquitously expressed. Research indicates:

• It is widely expressed across many tissue types

• Has notable expression in brain tissue

• Shows differential expression during cellular differentiation

• Expression levels can change during disease progression

When designing experiments involving H2AFY2, researchers should consider tissue-specific expression patterns and select appropriate control tissues. Western blot analysis has detected H2AFY2 in various cell lines including HepG2 cells, human liver tissue, and mouse kidney tissue , indicating its presence across different organ systems.

What criteria should be considered when selecting an H2AFY2 antibody for specific applications?

Selection of the appropriate H2AFY2 antibody requires careful consideration of multiple factors:

For example, if investigating H2AFY2 in human samples by Western blot, an antibody like the rabbit polyclonal anti-H2AFY2 (Center) has been validated for this application and shows reactivity with human samples . For multi-species studies, antibodies with cross-reactivity to human, mouse, and rat might be preferable .

How can researchers validate an H2AFY2 antibody's specificity and sensitivity?

Rigorous validation is crucial to ensure reliable results. A comprehensive validation protocol includes:

• Positive and negative controls:

  • Use cell lines known to express H2AFY2 (e.g., HepG2, MCF-7) as positive controls

  • Use knockout or knockdown cells as negative controls

• Multi-technique validation:

  • Confirm results across different applications (WB, IHC, IF)

  • Verify the observed molecular weight (~40 kDa) by Western blot

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Observe diminished or abolished signal

• Alternative antibodies:

  • Compare results with antibodies targeting different epitopes of H2AFY2

  • Consistent results across different antibodies increases confidence

• Immunoprecipitation followed by mass spectrometry:

  • Definitive validation of target specificity

For example, the H2AFY2 antibody from Proteintech (17030-1-AP) has been validated by Western blot in HepG2 cells, human liver tissue, and mouse kidney tissue, and by immunoprecipitation in HepG2 cells .

What are the key differences between polyclonal and monoclonal H2AFY2 antibodies in research applications?

The choice between polyclonal and monoclonal antibodies significantly impacts experimental outcomes:

What are the optimal conditions for using H2AFY2 antibodies in Western blotting?

Successful Western blotting with H2AFY2 antibodies requires optimization of several parameters:

• Sample preparation:

  • Nuclear extraction is recommended as H2AFY2 is a nuclear protein

  • Use specialized nuclear extraction buffers containing protease inhibitors

  • Typical loading amount: 35 μg/lane of total protein lysate

• Gel electrophoresis:

  • Use reducing conditions for optimal results

  • 10-12% SDS-PAGE gels are suitable for resolving the 40 kDa H2AFY2 protein

• Transfer conditions:

  • PVDF membrane is recommended

  • Transfer at 100V for 1-2 hours or 30V overnight at 4°C

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk or BSA in TBST

  • Primary antibody dilutions typically range from 1:500 to 1:2000

  • Incubate overnight at 4°C for optimal results

• Detection:

  • HRP-conjugated secondary antibodies work well

  • Expected band size: approximately 40 kDa

For example, when using the Proteintech H2AFY2 antibody (17030-1-AP), the recommended dilution is 1:500-1:2000 for Western blot applications .

What protocols are most effective for immunofluorescence detection of H2AFY2?

Optimized immunofluorescence protocol for H2AFY2 detection:

• Cell preparation:

  • Culture cells on coverslips to 70-80% confluence

  • PFA fixation (4%) for 10-15 minutes is recommended

  • Permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes

• Blocking and antibody incubation:

  • Block with 1-5% BSA or normal serum for 1 hour at room temperature

  • Incubate with primary antibody at 4 μg/ml for H2AFY2 antibodies

  • Incubate overnight at 4°C or 3 hours at room temperature

• Detection and imaging:

  • Use fluorophore-conjugated secondary antibodies (e.g., NorthernLights™ 557)

  • DAPI counterstaining for nuclear visualization

  • Mounting with anti-fade medium

• Image acquisition:

  • Confocal microscopy is recommended for precise nuclear localization

  • Use appropriate filters for the selected fluorophores

  • Adjust exposure to avoid saturation

H2AFY2 typically shows a nuclear speckled staining pattern, consistent with its role in chromatin organization. For example, in HeLa cells, specific staining of H2AFY2 is localized to the nuclei when using appropriate antibodies at concentrations around 25 μg/mL .

How should immunoprecipitation protocols be optimized for H2AFY2 research?

For successful immunoprecipitation of H2AFY2:

• Lysate preparation:

  • Use 1.0-3.0 mg of total protein lysate per IP reaction

  • Prepare nuclear extracts with high-salt buffers (300-450 mM NaCl)

  • Include protease inhibitors, phosphatase inhibitors, and deacetylase inhibitors

• Antibody amount and incubation:

  • Use 0.5-4.0 μg of antibody per IP reaction

  • Pre-clear lysate with protein A/G beads

  • Incubate antibody with lysate overnight at 4°C with gentle rotation

• Bead selection and washing:

  • For rabbit-derived antibodies, use protein A beads

  • For mouse-derived antibodies, use protein G beads

  • Wash 4-5 times with decreasing salt concentrations

• Elution and analysis:

  • Elute with SDS sample buffer at 95°C for 5 minutes

  • Analyze by Western blot to confirm successful IP

  • For downstream applications (e.g., mass spectrometry), consider native elution

The H2AFY2 antibody from Proteintech (17030-1-AP) has been successfully used for immunoprecipitation in HepG2 cells, demonstrating its effectiveness in capturing the native protein from cell lysates .

How can H2AFY2 antibodies be utilized in chromatin immunoprecipitation (ChIP) experiments?

ChIP protocols for H2AFY2 require special considerations due to its role in chromatin:

• Crosslinking and sonication:

  • Use 1% formaldehyde for 10 minutes at room temperature

  • Optimize sonication to achieve 200-500 bp DNA fragments

  • Test sonication efficiency by agarose gel electrophoresis

• Antibody selection and validation:

  • Use ChIP-validated H2AFY2 antibodies

  • Validate antibody using positive control regions known to contain H2AFY2

  • Include IgG negative controls and histone H3 positive controls

• Immunoprecipitation conditions:

  • Use 2-5 μg of antibody per ChIP reaction

  • Include protease inhibitors, phosphatase inhibitors

  • Incubate overnight at 4°C with rotation

• Analysis methods:

  • qPCR for targeted analysis of specific genomic regions

  • ChIP-seq for genome-wide profiling of H2AFY2 occupancy

  • Compare with datasets for other histone modifications

• Data interpretation:

  • H2AFY2 is expected to be enriched at transcriptionally repressed regions

  • Look for overlap with other heterochromatin marks (H3K9me3, H3K27me3)

  • Compare with expression data to correlate occupancy with gene silencing

For improved ChIP efficiency with histone variants like H2AFY2, consider using kits specifically designed for histone ChIP or high-sensitivity ChIP protocols .

What role does H2AFY2 play in epigenetic regulation and how can this be investigated using antibodies?

H2AFY2 contributes to epigenetic regulation through several mechanisms that can be investigated using specific antibody-based approaches:

• Transcriptional repression:

  • Use ChIP-seq with H2AFY2 antibodies to map genome-wide occupancy

  • Correlate with RNA-seq data to identify repressed genes

  • Perform sequential ChIP (re-ChIP) to identify co-occurrence with other repressive marks

• X chromosome inactivation:

  • Immunofluorescence with H2AFY2 antibodies to visualize enrichment on inactive X

  • Combined IF-FISH to simultaneously detect H2AFY2 and X chromosome

  • ChIP-seq to map H2AFY2 distribution across the X chromosome

• Interaction with histone modifications:

  • Co-IP experiments to identify interacting partners

  • Western blot with modification-specific antibodies after H2AFY2 IP

  • Mass spectrometry analysis of H2AFY2-containing nucleosomes

• Dynamic changes during development:

  • Time-course IF or Western blot analysis during differentiation

  • ChIP-seq at different developmental stages

  • Compare with other histone variant incorporation

Recent research has begun to elucidate the connection between histone variants like H2AFY2 and regulatory processes such as B-cell intrinsic regulation of antibody-mediated immunity, highlighting the importance of histone modification landscapes in immune responses .

How can researchers investigate the interplay between H2AFY2 and other histone modifications?

The functional relationship between H2AFY2 and other histone modifications represents an important area of epigenetic research:

• Sequential ChIP (re-ChIP) approach:

  • First ChIP with H2AFY2 antibody

  • Second ChIP with antibodies against specific histone modifications

  • Analysis reveals co-occurrence of H2AFY2 and modifications

• Proximity ligation assay (PLA):

  • Use antibodies against H2AFY2 and specific modifications

  • PLA signal indicates close proximity (<40 nm)

  • Quantify interactions in different cell types or conditions

• Mass spectrometry of H2AFY2-containing nucleosomes:

  • IP H2AFY2-containing nucleosomes

  • Perform mass spectrometry to identify associated modifications

  • Quantify modification enrichment compared to bulk nucleosomes

• Functional studies:

  • Combine H2AFY2 knockout/knockdown with ChIP-seq for various modifications

  • Analyze changes in modification patterns when H2AFY2 is depleted

  • Use enzyme inhibitors to block specific modifications and assess impact on H2AFY2 deposition

Research suggests connections between H2AFY2 and certain histone modifications. For example, the deubiquitinating enzyme BAP1 has been shown to regulate histone H2AK119ub levels, which impacts antibody-mediated immune responses , suggesting potential interplay between H2AFY2 and ubiquitination pathways in chromatin regulation.

What are the challenges in detecting post-translational modifications of H2AFY2 and how can they be overcome?

Detecting post-translational modifications (PTMs) of H2AFY2 presents several technical challenges:

• Low abundance of modified forms:

  • Enrich modified H2AFY2 using phospho-enrichment or ubiquitin-enrichment methods

  • Use larger starting material (10-20 mg of nuclear extract)

  • Consider label-free or immunoaffinity enrichment strategies

• Limited availability of modification-specific antibodies:

  • Develop custom antibodies against predicted modification sites

  • Validate using synthetic modified peptides

  • Use mass spectrometry as a complementary approach

• Dynamic and transient nature of modifications:

  • Add phosphatase inhibitors (e.g., sodium orthovanadate, β-glycerophosphate)

  • Include deacetylase inhibitors (e.g., TSA, sodium butyrate)

  • Use crosslinking approaches to capture transient interactions

• Detection strategy:

  • Two-dimensional gel electrophoresis to separate modified forms

  • Phos-tag™ gels for phosphorylated proteins

  • IP with H2AFY2 antibody followed by Western blot with modification-specific antibodies

• Analytical considerations:

  • Mass spectrometry with multiple fragmentation methods (ETD/HCD)

  • Include PTM-specific enrichment steps

  • Use targeted proteomics approaches for predicted modification sites

This methodological approach allows researchers to characterize the "histone code" associated with H2AFY2, gaining insights into how PTMs regulate its function in transcriptional repression and chromatin organization.

What are common issues in H2AFY2 antibody applications and how can they be resolved?

When working with H2AFY2 antibodies, researchers may encounter several challenges:

For optimal results with Western blot, consider using HeLa acid extract as a positive control for H2AFY2 detection . For immunofluorescence, PFA fixation followed by Triton X-100 permeabilization has shown good results with H2AFY2 antibodies .

How can researchers ensure reproducibility in experiments using H2AFY2 antibodies?

Ensuring reproducible results with H2AFY2 antibodies requires systematic quality control measures:

• Antibody validation and documentation:

  • Document catalog number, lot number, and source

  • Validate each new antibody lot with standard samples

  • Maintain detailed antibody validation records

• Standardized protocols:

  • Create detailed step-by-step protocols with precise reagent concentrations

  • Document incubation times and temperatures

  • Use consistent sample preparation methods

• Controls and standards:

  • Include positive controls (e.g., HepG2 cells, human liver tissue)

  • Use negative controls (IgG, non-expressing tissues)

  • Consider recombinant H2AFY2 as a standard

• Technical replicates and biological replicates:

  • Perform three technical replicates per experiment

  • Validate key findings across multiple biological samples

  • Consider inter-laboratory validation for critical findings

• Data analysis and reporting:

  • Use quantitative analysis methods

  • Report statistical methods and significance

  • Share detailed protocols with publications

Atlas Antibodies emphasizes that their H2AFY2 antibodies are manufactured using a standardized process to ensure rigorous quality levels, which helps maintain reproducibility between experiments .

What considerations are important when comparing results from different H2AFY2 antibody clones or sources?

When comparing results obtained using different H2AFY2 antibodies, researchers should consider:

• Epitope differences:

  • Antibodies targeting different regions (N-terminal, central, C-terminal) may give different results

  • Map the exact epitope sequences of each antibody

  • Consider how epitope location might affect detection of protein interactions

• Clonality and production methods:

  • Compare polyclonal versus monoclonal results carefully

  • Note differences in host species (rabbit vs. mouse)

  • Consider differences in immunization strategies (peptide vs. full protein)

• Validation parameters:

  • Review validation data for each antibody

  • Compare sensitivity and specificity measurements

  • Note differences in validated applications

• Cross-reactivity profiles:

  • Assess species cross-reactivity differences

  • Check for documented cross-reactivity with related proteins

  • Perform cross-validation experiments in the same samples

• Standardization approach:

  • Use the same positive controls across antibody comparisons

  • Standardize protocols as much as possible

  • Consider performing side-by-side comparisons

For instance, when comparing results between the RayBiotech anti-H2AFY2 (Center) antibody and the Proteintech H2AFY2 antibody , researchers should note that they target different regions of the protein, which might affect their ability to detect certain protein conformations or interactions.

How is H2AFY2 being investigated in the context of disease mechanisms and biomarker research?

Recent research has begun exploring H2AFY2's role in various disease contexts:

• Cancer research:

  • Altered H2AFY2 expression in certain cancer types

  • Potential role in silencing tumor suppressor genes

  • Methodological approach: Compare H2AFY2 levels in tumors vs. normal tissue by IHC and Western blot

• Neurodevelopmental disorders:

  • H2AFY2's role in brain development suggests connections to neurodevelopmental conditions

  • Investigate through animal models and patient samples

  • Use IF to examine H2AFY2 distribution in neural tissues

• Immune system regulation:

  • Recent research connects histone regulation to antibody-mediated immunity

  • Study H2AFY2 in B cell differentiation and antibody production

  • Combine ChIP-seq and RNA-seq in immune cell populations

• Aging and epigenetic reprogramming:

  • Changes in heterochromatin markers like H2AFY2 during aging

  • Correlation with age-related gene expression changes

  • Time-course studies with quantitative IF and Western blot

• Biomarker development:

  • Potential use of H2AFY2 as a biomarker for specific conditions

  • Develop sensitive detection methods in accessible samples

  • Validate across multiple patient cohorts

A 2024 study demonstrated that BAP1 plays a critical role in regulating histone H2A ubiquitination levels, which impacts B-cell function and antibody-mediated immunity . This suggests potential connections between H2AFY2 regulation and immune system disorders.

What new methodological approaches are being developed for studying H2AFY2 in chromatin dynamics?

Innovative techniques for investigating H2AFY2's role in chromatin:

• CUT&RUN and CUT&Tag techniques:

  • Provide higher resolution mapping of H2AFY2 than traditional ChIP

  • Require less starting material

  • Can be performed in single cells

• Live-cell imaging of H2AFY2:

  • CRISPR-based tagging of endogenous H2AFY2

  • Super-resolution microscopy to visualize distribution

  • Live tracking of dynamics during cell cycle

• Proximity labeling approaches:

  • BioID or APEX2 fusion with H2AFY2

  • Identify proteins in close proximity in living cells

  • Map the H2AFY2 interaction network

• Single-cell multi-omics:

  • Combined single-cell ATAC-seq and RNA-seq

  • Correlate H2AFY2 occupancy with gene expression

  • Identify cell-type specific patterns

• Cryo-EM studies:

  • Structural analysis of H2AFY2-containing nucleosomes

  • Compare with canonical nucleosomes

  • Investigate structural impacts on chromatin fiber

These approaches provide higher resolution insights into H2AFY2 function than traditional antibody-based methods alone, often complementing classical approaches. For example, combining H2AFY2 ChIP-seq data with CUT&Tag data could provide more comprehensive mapping of genome-wide distribution patterns.

How do researchers investigate the functional differences between H2AFY2 and other macroH2A variants?

Distinguishing the specific functions of H2AFY2 from other macroH2A variants requires specialized approaches:

• Isoform-specific knockdown/knockout:

  • Design siRNAs or CRISPR guides specific to H2AFY2

  • Validate specificity by Western blot using variant-specific antibodies

  • Perform rescue experiments with individual variants

• Variant-specific ChIP-seq:

  • Use antibodies that specifically recognize H2AFY2 vs. H2AFY1

  • Compare genome-wide distribution patterns

  • Identify unique and shared target sites

• Domain swap experiments:

  • Create chimeric proteins between H2AFY2 and other variants

  • Express in knockout backgrounds

  • Determine which domains confer specific functions

• Developmental and tissue-specific expression:

  • Compare expression patterns across tissues and developmental stages

  • Use variant-specific antibodies in Western blot and IHC

  • Correlate with functional differences between tissues

• Interaction partner identification:

  • Perform co-IP with variant-specific antibodies

  • Identify specific binding partners by mass spectrometry

  • Validate interactions with co-IP and proximity ligation assay

For example, the MacroH2A1.2 Antibody from Cell Signaling Technology specifically detects MacroH2A1.2 (isoform 2) and can be used in Western blot and immunofluorescence to compare its expression and localization with H2AFY2 .

What databases and resources are available for researching H2AFY2 function and interactions?

Researchers investigating H2AFY2 can leverage numerous specialized resources:

• Protein databases and tools:

  • UniProt (Q9P0M6 for human H2AFY2): Comprehensive protein information

  • Protein Data Bank (PDB): 3D structural data (e.g., PDB:2XD7)

  • STRING database: Protein-protein interaction networks

• Genomic resources:

  • UCSC Genome Browser: Visualize H2AFY2 gene and conservation

  • ENCODE Project: ChIP-seq and other functional genomics data

  • 4D Nucleome Project: Chromatin structure information

• Epigenomic databases:

  • Roadmap Epigenomics: Tissue-specific epigenetic marks

  • IHEC Data Portal: International Human Epigenome Consortium data

  • ChIP-Atlas: Collection of published ChIP-seq experiments

• Expression databases:

  • GTEx Portal: Tissue-specific expression data

  • Human Protein Atlas: Protein expression across tissues

  • Single Cell Expression Atlas: Cell-type specific expression

• Research reagents:

  • Addgene: Plasmids for H2AFY2 expression (e.g., plasmid #39052)

  • Antibody validation databases: Antibodypedia, Antibody Registry

  • Knockout/knockdown resources: CRISPR/RNAi libraries

The H2AFY2 gene has the NCBI Gene ID 55506 and various research resources including plasmids are available for investigating its function .

How can researchers design effective control experiments when studying H2AFY2?

Robust control strategies for H2AFY2 research:

• Antibody controls:

  • Isotype controls (matched IgG) for immunoprecipitation and ChIP

  • Pre-immune serum controls for polyclonal antibodies

  • Peptide competition assays to verify specificity

• Expression controls:

  • Positive control tissues/cells (HepG2, human liver)

  • Knockdown/knockout validation

  • Rescue with exogenous expression

• Experimental design controls:

  • Include multiple antibodies targeting different epitopes

  • Use both polyclonal and monoclonal antibodies when possible

  • Include biological and technical replicates

• Cross-validation approaches:

  • Verify key findings with orthogonal techniques

  • Compare antibody-based results with genomic methods

  • Validate across multiple model systems

• Recombinant protein controls:

  • Use recombinant H2AFY2 as positive control

  • Create standard curves for quantitative assays

  • Include spike-in controls for recovery assessment

Researchers can utilize recombinant H2AFY2 proteins as controls, such as those available through plasmid repositories like Addgene (plasmid #39052) , which can be expressed and purified for use as positive controls in assays.

What are emerging areas of research involving H2AFY2 in cellular reprogramming and differentiation?

H2AFY2's role in cellular identity and fate determination represents an exciting frontier:

• Stem cell differentiation:

  • Monitor H2AFY2 incorporation during differentiation pathways

  • Perform ChIP-seq at multiple timepoints

  • Correlate with expression changes of developmental genes

• Cellular reprogramming:

  • Track H2AFY2 redistribution during iPSC generation

  • Investigate its role in maintaining epigenetic barriers

  • Test if H2AFY2 manipulation enhances reprogramming efficiency

• Tissue regeneration models:

  • Study H2AFY2 dynamics during regeneration processes

  • Compare with development and homeostasis

  • Target H2AFY2 to enhance regenerative capacity

• Aging and senescence:

  • Investigate age-associated changes in H2AFY2 distribution

  • Correlation with senescence-associated heterochromatin

  • Potential role in age-related epigenetic drift

• Transgenerational epigenetic inheritance:

  • H2AFY2's potential role in transmitting epigenetic information

  • Analysis in gametes and early embryonic development

  • Connections to environmental response mechanisms

The connection between H2AFY2 and cellular differentiation pathways, particularly in brain development , suggests important roles in cell fate decisions that warrant further investigation using the antibody-based approaches outlined in this document.

How might advances in technology enhance our ability to study H2AFY2 function in the future?

Technological innovations are poised to transform H2AFY2 research:

• Advanced imaging techniques:

  • Super-resolution microscopy for detailed chromatin structure

  • STORM/PALM imaging of individual H2AFY2-containing nucleosomes

  • Live-cell tracking with minimal interference

• Single-cell multi-omics integration:

  • Combined ChIP-seq, ATAC-seq, and RNA-seq at single-cell level

  • Spatial transcriptomics with H2AFY2 mapping

  • New computational methods for integrated analysis

• Engineered antibody technologies:

  • Nanobodies against H2AFY2 for improved nuclear penetration

  • Split-fluorescent protein complementation for interaction studies

  • Engineered antibody fragments for super-resolution applications

• CRISPR-based epigenome editing:

  • Targeted recruitment or removal of H2AFY2 at specific loci

  • Precise manipulation of chromatin domains

  • Study causality rather than correlation

• AI-enhanced analysis:

  • Machine learning to identify subtle H2AFY2 distribution patterns

  • Predictive modeling of H2AFY2 function

  • Automated image analysis for high-throughput screening

These technological advances will complement traditional antibody-based approaches, providing more precise and comprehensive insights into H2AFY2 biology than previously possible.

What challenges remain in understanding the structural biology of H2AFY2 and its impact on chromatin organization?

Despite progress, significant challenges remain in understanding H2AFY2 structure and function:

• High-resolution structural studies:

  • Obtaining crystal or cryo-EM structures of complete H2AFY2 nucleosomes

  • Understanding the macro domain's interaction with chromatin

  • Determining structural transitions during chromatin folding

• Dynamic structural changes:

  • Capturing conformational changes during regulatory events

  • Understanding impact on higher-order chromatin structure

  • Developing tools to study dynamics in living cells

• Locus-specific functions:

  • Determining why H2AFY2 targets specific genomic regions

  • Identifying sequence-specific recruitment mechanisms

  • Developing methods for locus-specific analysis

• Interactome complexity:

  • Comprehensive mapping of H2AFY2 interaction partners

  • Cell-type and context-specific interactions

  • Structural basis of protein-protein interactions

• Integration with other chromatin features:

  • Relationship with DNA methylation

  • Coordination with histone modifications

  • Impact on chromatin accessibility and organization