HIST1H2AG (Ab-119) Antibody

What is HIST1H2AG and what cellular processes is it involved in?

HIST1H2AG is a core component of the nucleosome, functioning as a histone H2A type 1 protein. It plays a central role in chromatin structure by helping to wrap and compact DNA, which regulates DNA accessibility to cellular machinery. Through this function, HIST1H2AG is critically involved in transcription regulation, DNA repair, DNA replication, and maintaining chromosomal stability. The accessibility of DNA is regulated through post-translational modifications of histones (often referred to as the "histone code") and nucleosome remodeling processes .

As a core histone protein, HIST1H2AG is fundamentally involved in the formation of the nucleosome octamer around which DNA wraps approximately 1.7 times. This organization helps compact the DNA and plays a crucial role in epigenetic regulation by determining which genes are accessible for transcription.

What are the key applications for HIST1H2AG (Ab-119) Antibody in experimental settings?

The HIST1H2AG (Ab-119) Antibody can be utilized in multiple experimental applications that allow researchers to investigate histone biology and chromatin structure:

• Chromatin Immunoprecipitation (ChIP): For identifying DNA binding sites and analyzing protein-DNA interactions

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of HIST1H2AG

• Immunofluorescence (IF): For visualizing cellular localization of HIST1H2AG

• Immunohistochemistry (IHC): For detecting HIST1H2AG in tissue sections

This versatility makes the antibody valuable for diverse experimental approaches when studying histone function and chromatin dynamics.

How should HIST1H2AG (Ab-119) Antibody be stored and handled to maintain its efficacy?

Proper storage and handling of the HIST1H2AG (Ab-119) Antibody is crucial for maintaining its binding specificity and sensitivity. Upon receipt, the antibody should be stored at either -20°C or -80°C. Researchers should avoid repeated freeze-thaw cycles as these can degrade antibody quality .

The antibody is supplied in liquid form with a specific diluent buffer composition:

• 0.03% Proclin 300 (preservative)

• 50% Glycerol

• 0.01M PBS at pH 7.4

For optimal results, aliquot the antibody upon first thaw to minimize freeze-thaw cycles. When preparing working dilutions, use freshly prepared buffers and work in a clean environment to prevent contamination.

What is the specificity of HIST1H2AG (Ab-119) Antibody and how does it compare to antibodies for other H2A variants?

The HIST1H2AG (Ab-119) Antibody is a rabbit polyclonal antibody generated against a peptide sequence around lysine 119 derived from human Histone H2A type 1 . This specificity is important because although there are multiple histone H2A variants with high sequence similarity, they can have distinct functions.

The antibody specifically recognizes HIST1H2AG, which is one of several H2A variants in the human genome. When selecting between different H2A antibodies, researchers should consider that:

• HIST1H2AG (Ab-119) is derived from immunization with a peptide specifically surrounding the Lys 119 region

• It's important to note that some H2A genes (including HIST1H2AB, 2AC, 2AD, 2AE, 2AG, 2AI, 2AN, and 2AO) encode identical proteins despite having different expression patterns

• For experiments requiring high specificity, validation using knockout controls or peptide competition is recommended

How can HIST1H2AG (Ab-119) Antibody be optimized for Chromatin Immunoprecipitation (ChIP) experiments?

For optimal ChIP results with HIST1H2AG (Ab-119) Antibody:

Researchers studying H2A modifications, particularly those near lysine 119, should consider that this antibody's epitope may be influenced by post-translational modifications. For example, when studying H2AK119 ubiquitination, which is a critical modification deposited by Polycomb Repressive Complex 1, this specific antibody may have reduced binding if the target lysine is modified .

What are the recommended protocols for using HIST1H2AG (Ab-119) Antibody in immunofluorescence studies?

For optimal immunofluorescence results with HIST1H2AG (Ab-119) Antibody:

• Fixation: Use 4% paraformaldehyde for 10-15 minutes at room temperature.

• Permeabilization: Treat with 0.2% Triton X-100 for 5-10 minutes to allow antibody access to nuclear proteins.

• Antigen retrieval: This is particularly important for detecting nuclear antigens like histones.

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • 10-20 minutes at 95-100°C

• Blocking: Block with 5% normal serum (from the species of the secondary antibody) for 1 hour.

• Primary antibody incubation:

  • Starting dilution: 1:100-1:500

  • Incubate overnight at 4°C in a humidified chamber

  • Optimize concentration based on signal-to-noise ratio

• Controls:

  • Negative control: omit primary antibody

  • Comparison with another validated H2A antibody

• Signal detection: Use appropriate fluorophore-conjugated secondary antibodies with minimal cross-reactivity.

• Counterstaining: DAPI for nuclear visualization helps confirm the expected nuclear localization pattern of HIST1H2AG.

When interpreting results, remember that histone staining should show clear nuclear localization with potentially variable intensity depending on cell cycle stage and transcriptional activity.

How should western blot protocols be adapted for optimal detection of HIST1H2AG?

Detecting histone proteins by western blot requires specific adaptations:

• Extraction methods:

  • Use specialized histone extraction protocols (acid extraction)

  • Commercial histone extraction kits often yield better results than standard RIPA buffer extractions

• Gel selection:

  • 15-18% SDS-PAGE gels are recommended for resolving small histone proteins

  • HIST1H2AG is approximately 14 kDa

• Transfer conditions:

  • Use PVDF membrane (0.2 μm pore size)

  • Methanol-containing transfer buffer improves histone protein binding

  • Transfer at lower voltage for longer time (30V overnight)

• Blocking:

  • 5% BSA in TBST is generally more effective than milk-based blocking buffers

• Antibody dilution:

  • Start with 1:1000 dilution and optimize

  • Incubate overnight at 4°C

• Loading control selection:

  • Traditional housekeeping genes may not be ideal

  • Total histone H3 or H4 can serve as appropriate loading controls for nuclear proteins

• Expected band size:

  • Unmodified HIST1H2AG: ~14 kDa

  • Post-translationally modified forms may show altered migration

A critical consideration is that histone proteins are highly conserved, making appropriate controls essential to confirm specificity.

How does expression of HIST1H2AG compare to other histone H2A variants during the cell cycle?

Expression patterns of histone H2A variants show significant differences during the cell cycle, particularly between replication-dependent and replication-independent variants:

HIST1H2AG follows a replication-dependent expression pattern typical of canonical histones. Studies in mouse models have shown that HIST1H2AG (Hist1h2ag) expression increases from the beginning of S-phase, reaches its peak in mid-S-phase (2-4 hours into S-phase), and then decreases toward the end of S-phase (6 hours) .

Comparative expression analysis reveals striking differences in expression levels between different H2A genes:

• The expression level of Hist1h2ae is 10-30 times higher than that of Hist1h2ag

• Among the 13 genes in the Hist1 cluster, Hist1h2ae expression is approximately 100 times that of Hist1h2aa

• Among genes with identical protein products, significant expression differences exist

What role does Lysine 119 of histone H2A play in chromatin regulation and how can the HIST1H2AG (Ab-119) Antibody be used to study these processes?

Lysine 119 of histone H2A (H2AK119) is a critical site for post-translational modification, particularly ubiquitination, which has significant implications for gene regulation:

• H2AK119 Ubiquitination:

  • H2AK119Ub is deposited by Polycomb Repressive Complex 1 (PRC1)

  • This modification is associated with gene silencing and is crucial in developmental gene regulation

  • H2AK119Ub is deubiquitinated by the Polycomb Repressive Deubiquitinase complex (PR-DUB)

• Role in Cancer:

  • Dysregulation of H2AK119Ub is implicated in cancer development

  • The PR-DUB subunits BAP1 and ASXL1 are among the most frequently mutated epigenetic factors in human cancers

• Structural Insights:

  • Recent cryo-EM structures reveal how PR-DUB specifically recognizes and deubiquitinates H2AK119Ub

  • This specificity involves molecular interactions between BAP1 and ASXL1 with histones and DNA, which restructure the nucleosome

The HIST1H2AG (Ab-119) Antibody can be utilized to study these processes through:

• ChIP experiments to map genome-wide distribution of HIST1H2AG

• Combining with antibodies against ubiquitin to study H2AK119Ub levels

• Immunoprecipitation followed by mass spectrometry to identify interaction partners

• Monitoring changes in H2A localization in response to treatments affecting Polycomb complexes

When studying H2AK119 modifications, researchers should note that this antibody's epitope includes Lysine 119, so binding may be affected when this residue is modified.

How can HIST1H2AG (Ab-119) Antibody be used in multi-parameter flow cytometry to study cell cycle-dependent histone dynamics?

For multi-parameter flow cytometry applications studying cell cycle-dependent HIST1H2AG dynamics:

• Sample preparation protocol:

  • Fix cells in 70-80% ethanol or methanol (dropwise while vortexing)

  • Permeabilize with 0.25% Triton X-100 for intranuclear access

  • Use specialized buffers for histone detection that maintain nuclear integrity

• Antibody panel design:

  • HIST1H2AG (Ab-119) Antibody (detected with appropriate fluorophore-conjugated secondary)

  • DNA content marker (DAPI or propidium iodide)

  • S-phase marker (anti-BrdU or EdU with click chemistry)

  • Mitotic marker (phospho-histone H3 Ser10)

  • Other histone variants or modifications of interest

• Controls:

  • Single-stained controls for compensation

  • FMO (Fluorescence Minus One) controls

  • Isotype controls for each antibody

• Analysis strategy:

  • Gate on single cells using forward/side scatter and pulse width parameters

  • Define cell cycle phases based on DNA content

  • Analyze HIST1H2AG signal intensity across cell cycle phases

  • Compare with other histone variants/modifications

This approach allows for quantitative analysis of how HIST1H2AG levels change during the cell cycle, which can be correlated with the replication-dependent expression pattern observed in gene expression studies .

What are the analytical considerations when studying the relationship between HIST1H2AG and chromatin remodeling complexes?

When investigating interactions between HIST1H2AG and chromatin remodeling complexes:

• Co-immunoprecipitation approaches:

  • Use specialized nuclear extraction buffers that preserve protein-protein interactions

  • Consider crosslinking to capture transient interactions

  • Include appropriate negative controls (IgG, unrelated nuclear protein)

  • Western blot or mass spectrometry can identify interaction partners

• Sequential ChIP (Re-ChIP):

  • To determine co-occupancy of HIST1H2AG with chromatin remodelers

  • First IP with HIST1H2AG (Ab-119) Antibody

  • Elute and perform second IP with antibodies against remodeling components

• Proximity ligation assay:

  • For visualizing protein-protein interactions in situ

  • Requires additional antibody against predicted interaction partner

  • Signals only appear when proteins are in close proximity

• Analytical challenges:

  • Distinguishing direct from indirect interactions

  • Accounting for cell cycle variation in histone content

  • Determining causality in observed associations

Researchers should consider that HIST1H2AG interactions may be influenced by SWR1-type chromatin remodeling complexes, which are known to mediate the deposition of histone variants into chromatin . Though most research on SWR1 complexes has focused on H2A.Z deposition, the mechanisms may be relevant for understanding canonical H2A dynamics as well.

What are common troubleshooting approaches for non-specific binding when using HIST1H2AG (Ab-119) Antibody?

When encountering non-specific binding with HIST1H2AG (Ab-119) Antibody:

• For Western Blot applications:

  • Increase blocking time and concentration (try 5% BSA for 2 hours)

  • Increase washing stringency (0.1% to 0.3% Tween-20)

  • Reduce primary antibody concentration

  • Add competing proteins to antibody diluent (0.1-0.5% BSA)

  • Pre-absorb antibody with cell/tissue lysate from non-relevant species

• For Immunohistochemistry/Immunofluorescence:

  • Include additional blocking steps with 10% serum

  • Use avidin/biotin blocking if using biotin-based detection systems

  • Optimize fixation conditions (overfixation can increase background)

  • Use detergents in wash buffers (0.1-0.3% Triton X-100)

• For ChIP applications:

  • Increase pre-clearing time with protein A/G beads

  • Add competitor DNA (sonicated salmon sperm DNA)

  • Optimize crosslinking conditions

  • Increase washing stringency with higher salt concentrations

• General considerations:

  • Quality of blocking reagents can significantly impact background

  • Temperature of incubations (4°C typically reduces non-specific interactions)

  • Freshness of reagents (especially detection systems)

When troubleshooting, always include appropriate negative controls and consider testing multiple batches of the antibody if possible.

How can researchers distinguish between different histone H2A subtypes when their protein sequences are identical?

Distinguishing between histone H2A subtypes with identical protein sequences presents a significant challenge. Since several H2A genes (including HIST1H2AB, 2AC, 2AD, 2AE, 2AG, 2AI, 2AN, and 2AO) encode identical proteins , antibody-based approaches cannot differentiate between them. Researchers can employ the following strategies:

• mRNA expression analysis:

  • Design primers specific to unique untranslated regions of each H2A gene

  • Use quantitative RT-PCR to measure expression levels

  • RNA-Seq with appropriate bioinformatic pipelines for isoform quantification

• Promoter-specific analysis:

  • ChIP assays targeting transcription factors specific to different H2A promoters

  • Analysis of promoter methylation or histone modifications

• Reporter gene constructs:

  • Generate constructs with promoters from different H2A genes

  • Measure activity under different conditions to infer endogenous regulation

• Advanced proteomic approaches:

  • While protein sequences may be identical, post-translational modifications might differ

  • Targeted mass spectrometry may detect subtle differences in modification patterns

Researchers studying histone H2A variants should carefully design experiments to address this limitation. For instance, comparative studies have shown that despite identical protein sequences, the expression level of Hist1h2ae is 10-30 times that of Hist1h2ag in mouse models , suggesting distinct regulatory mechanisms.

What considerations should be made when using HIST1H2AG (Ab-119) Antibody across different model organisms?

When using HIST1H2AG (Ab-119) Antibody across different model organisms:

• Sequence homology assessment:

  • The antibody is raised against human Histone H2A peptide sequence

  • Check protein sequence alignment between human HIST1H2AG and target organism

  • Particularly examine conservation around the Lys119 region

• Validation requirements:

  • Western blot validation in each new species

  • Positive and negative controls specific to each organism

  • Consider knockout/knockdown controls when available

• Species-specific considerations:

  Species	Sequence Homology	Expected Cross-Reactivity	Validation Needed
  Mouse	Very high	Likely	Western blot
  Rat	Very high	Likely	Western blot
  Zebrafish	High	Possible	Extensive
  Drosophila	Moderate	Limited	Extensive
  C. elegans	Moderate	Limited	Extensive
  Plants	Low	Unlikely	Not recommended

• Application adjustments:

  • Protocol modifications may be necessary for different organisms

  • Extraction methods may need optimization

  • Fixation and permeabilization conditions may differ

• Alternative approaches:

  • For organisms with low homology, consider using species-specific antibodies

  • Mass spectrometry-based approaches may be more reliable for cross-species studies

  • Consider epitope-tagged constructs for non-mammalian systems

Researchers should note that while core histones are highly conserved across eukaryotes, subtle sequence differences exist that may affect antibody recognition.

How can HIST1H2AG (Ab-119) Antibody be utilized in studying the relationship between histone variants and cancer progression?

The HIST1H2AG (Ab-119) Antibody can be valuable in cancer research through:

• Tissue microarray analysis:

  • Compare HIST1H2AG levels across tumor grades and types

  • Correlate with patient outcomes and treatment responses

  • Combine with markers of cell proliferation and differentiation

• Chromatin landscape characterization:

  • ChIP-seq to map genome-wide distribution in cancer vs. normal cells

  • Integrate with transcriptomic data to correlate with gene expression changes

  • Analyze co-localization with cancer-specific transcription factors

• Epigenetic modification patterns:

  • Since H2AK119 is a critical site for ubiquitination by PRC1

  • Changes in ubiquitination patterns may drive cancer progression

  • The BAP1 and ASXL1 components of PR-DUB are frequently mutated in human cancers

• Methodological approach:

  • Cell line panels representing cancer progression

  • Patient-derived xenografts

  • Tissue samples with appropriate controls

  • Multi-parameter analysis (combining with other markers)

• Technical considerations:

  • Tissue fixation affects histone epitope accessibility

  • Antigen retrieval optimization is critical

  • Controls should include normal adjacent tissue

This research direction is particularly relevant given the findings that components of histone modification machinery targeting H2AK119 are frequently altered in cancer, with over 50 mutations in BAP1 and ASXL1 identified that can dysregulate H2AK119Ub deubiquitination .

What are the considerations for using HIST1H2AG (Ab-119) Antibody in high-resolution microscopy techniques?

For applying HIST1H2AG (Ab-119) Antibody in high-resolution microscopy:

• Super-resolution techniques compatibility:

  • STORM/PALM: Requires photoswitchable fluorophores conjugated to secondary antibodies

  • STED: Works with conventional fluorophores but requires high photostability

  • SIM: Less demanding on fluorophore properties

• Sample preparation optimization:

  • Fixation: Mild fixation (2% PFA) often preserves epitope accessibility

  • Chromatin accessibility: Consider mild permeabilization with digitonin

  • Non-specific binding: Critical to minimize for high signal-to-noise ratio

  • Mounting media: Use specialized media for super-resolution techniques

• Technical considerations:

  • Primary antibody concentration: Usually lower than conventional microscopy

  • Incubation time: Extended incubation (24-48h at 4°C) improves penetration

  • Blocking: More stringent to reduce background

  • Secondary antibody selection: High-quality, minimal cross-reactivity

• Controls and validation:

  • Resolution standards to ensure system performance

  • Multiple imaging approaches to confirm structures

  • Co-localization with known nuclear landmarks

• Analysis approaches:

  • Quantitative analysis of HIST1H2AG distribution patterns

  • Correlation with chromatin density markers

  • 3D reconstruction of nuclear organization

High-resolution imaging can provide insights into the spatial organization of HIST1H2AG within the nucleus and its relationship to chromatin domains and nuclear architecture.

How can researchers integrate HIST1H2AG antibody-based approaches with genomic and transcriptomic analyses?

Integrating HIST1H2AG antibody-based methods with genomic and transcriptomic techniques:

• ChIP-seq and CUT&RUN approaches:

  • Map genome-wide distribution of HIST1H2AG

  • Integrate with RNA-seq data to correlate with gene expression patterns

  • Compare with other histone variant distributions and modifications

  • Analyze relationship with chromatin accessibility (ATAC-seq)

• Sequential methodologies:

  • ChIP-seq followed by RNA-seq from the same sample

  • Cell sorting based on HIST1H2AG levels followed by genomic analyses

  • Single-cell approaches combining protein and RNA detection

• Data integration strategies:

  • Correlation analysis between HIST1H2AG binding and gene expression

  • Machine learning approaches to identify predictive patterns

  • Network analysis to identify regulatory hubs

• Analytical considerations:

  Data Type	Analysis Approach	Integration Method	Challenge
  ChIP-seq	Peak calling, motif analysis	Overlap with transcription sites	Peak width variation
  RNA-seq	Differential expression	Correlation with ChIP signal	Indirect effects
  ATAC-seq	Accessibility quantification	Co-occurrence analysis	Resolution differences
  Hi-C	Topological domain identification	Enrichment in domains	Complex data structure

• Biological interpretation frameworks:

  • Cell cycle-dependent analysis to account for replication-dependent expression

  • Developmental stage comparisons

  • Treatment response studies

This integrated approach can provide comprehensive insights into how HIST1H2AG contributes to gene regulation and chromatin architecture in different biological contexts.