HIST1H2AG (Ab-36) Antibody

What is the specificity of the HIST1H2AG (Ab-36) antibody?

The HIST1H2AG (Ab-36) antibody is a polyclonal antibody raised in rabbits against a peptide sequence around the lysine 36 residue derived from Human Histone H2A type 1 . This antibody specifically recognizes the histone H2A type 1 protein (also known as H2A.1) in both human and mouse samples . The antibody targets a region that is part of the core structure of histone H2A, allowing detection of this essential nucleosome component. Specifically, this antibody corresponds to the UniProt ID P0C0S8 .

What experimental applications has this antibody been validated for?

The HIST1H2AG (Ab-36) antibody has been validated for several common laboratory techniques:

This antibody has been successfully used in analyzing mouse heart tissue by Western blot and human glioma tissue by immunohistochemistry . Validation data demonstrates specific staining patterns consistent with histone localization in HeLa cells using immunofluorescence techniques .

How should I optimize HIST1H2AG (Ab-36) antibody for chromatin immunoprecipitation (ChIP) experiments?

While not explicitly listed among the validated applications in all sources, antibodies against histone proteins are frequently used in ChIP experiments. For optimal ChIP performance with the HIST1H2AG (Ab-36) antibody:

• Cross-linking conditions: Standard 1% formaldehyde for 10 minutes at room temperature is generally sufficient for histone proteins.

• Sonication parameters: Aim for chromatin fragments between 200-500 bp for optimal histone detection.

• Antibody concentration: Begin with 2-5 μg of antibody per ChIP reaction and optimize if needed.

• Beads selection: Protein A/G beads are compatible with rabbit IgG antibodies.

• Washing stringency: Include high-salt washes (up to 300-500 mM NaCl) to reduce non-specific binding.

Research has shown that histone ChIP experiments benefit from evaluating extraction efficiency to ensure proper chromatin preparation . Similar to methods used for H3.1 and H3.3 ChIP assays, validation of specificity can be performed by comparing RhIP-ChIP (recombinant histone immunoprecipitation-ChIP) patterns with endogenous histone ChIP patterns .

What controls should I use when working with HIST1H2AG (Ab-36) antibody?

Proper experimental controls are essential for valid interpretation of results:

• Negative controls:

  • IgG control: Use rabbit IgG at the same concentration as the primary antibody

  • No-antibody control: Perform the procedure omitting the primary antibody

  • Peptide competition: Pre-incubate the antibody with excess immunizing peptide

• Positive controls:

  • Known positive tissue samples: Human glioma tissue or HeLa cells have been validated

  • Recombinant HIST1H2AG protein: Can verify antibody specificity

  • Cell types with high histone expression: Rapidly dividing cells typically have high histone levels

• Knockdown/knockout validation:

  • siRNA against HIST1H2AG: Similar to approaches used for other histone variants

  • Comparison with other H2A antibodies: To confirm specificity for the H2A.1 variant

How can this antibody be used to study the relationship between HIST1H2AG and RNA binding?

Recent research has revealed intriguing connections between histone variants and RNA binding. While H2A.B (a different H2A variant) has been directly implicated in RNA binding , the potential RNA interactions of HIST1H2AG remain less explored. To investigate potential RNA interactions:

• RNA immunoprecipitation (RIP): Using HIST1H2AG (Ab-36) antibody to pull down protein-RNA complexes, followed by RNA-seq analysis.

• Crosslinking immunoprecipitation (CLIP): To identify direct RNA-protein interactions involving HIST1H2AG.

• Comparative studies: Between HIST1H2AG and H2A.B binding patterns could elucidate functional differences.

Research on H2A.B has shown that histone-RNA interactions can be inhibited by the presence of endogenous RNA, suggesting competitive binding mechanisms . Similar experiments could determine if HIST1H2AG exhibits comparable RNA binding properties.

How does HIST1H2AG differ from other H2A variants, and can this antibody distinguish between them?

Histone H2A has multiple variants with distinct functions in chromatin regulation:

The evolutionary conservation of histone proteins means that the antibody shows cross-reactivity between human and mouse samples , but researchers should verify specificity against the particular H2A variant of interest in their experimental system.

Western Blot Protocol Optimization:

• Sample preparation:

  • Extract histones using acid extraction (0.2N HCl) for enrichment

  • Use histone-optimized lysis buffers containing deacetylase inhibitors (e.g., sodium butyrate)

  • Recommended protein amount: 10-30 μg per lane

• Gel electrophoresis:

  • Use 15-18% SDS-PAGE gels for optimal separation of low molecular weight histones

  • Run at lower voltage (80-100V) for better resolution

• Transfer conditions:

  • PVDF membrane (0.2 μm pore size) recommended for small proteins

  • Transfer in 25mM Tris, 192mM glycine, 20% methanol at 30V overnight (4°C)

• Antibody incubation:

  • Block with 5% BSA in TBST (not milk, which contains histones)

  • Dilute primary antibody 1:500-5000 in 5% BSA/TBST

  • Incubate overnight at 4°C with gentle agitation

Immunofluorescence Protocol:

• Fixation:

  • 4% paraformaldehyde for 10-15 minutes

  • Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

• Antibody application:

  • Dilute antibody 1:50-200 in antibody dilution buffer

  • Incubate overnight at 4°C in humidified chamber

  • Use appropriate fluorophore-conjugated secondary antibody

• Nuclear counterstain:

  • DAPI (1 μg/ml) for 5 minutes

  • Mount with anti-fade mounting medium

How can I distinguish technical artifacts from true signal when using this antibody?

Common technical issues and resolutions include:

• High background:

  • Increase blocking time or concentration

  • Reduce primary antibody concentration

  • Add additional washing steps with higher salt concentration (up to 300mM NaCl)

  • Use 0.03% Proclin 300 as a preservative to maintain antibody quality

• Weak or no signal:

  • Ensure sample preparation preserves histone epitopes

  • Optimize antigen retrieval for IHC (citrate buffer, pH 6.0)

  • Increase antibody concentration or incubation time

  • Check storage conditions (maintain at -20°C in small aliquots)

• Non-specific bands in Western blot:

  • Perform peptide competition assay

  • Compare with other H2A antibodies targeting different epitopes

  • Use gradient gels for better resolution

• Validating true signals:

  • Compare staining patterns with published histone localization data

  • Use siRNA knockdown of HIST1H2AG as a negative control

  • Evaluate correlation with other histone marks or protein co-localization

What role does HIST1H2AG play in cancer research, and how can this antibody contribute?

Recent studies have highlighted the importance of histone variants and mutations (oncohistones) in cancer development . While HIST1H2AG-specific roles in cancer have not been extensively documented in the provided search results, research approaches using this antibody could include:

• Expression analysis:

  • Compare HIST1H2AG levels between normal and cancer tissues

  • Correlate expression with clinical outcomes or cancer subtypes

• Histone modification studies:

  • Investigate how post-translational modifications on HIST1H2AG differ in cancer

  • Examine co-localization with oncogenic histone variants

• Nucleosome positioning:

  • Analyze HIST1H2AG distribution in cancer-associated genomic regions

  • Study changes in chromatin accessibility related to HIST1H2AG incorporation

Research has shown that histone mutations can function as oncogenic drivers, particularly in pediatric tumors . The HIST1H2AG (Ab-36) antibody could be used to investigate whether changes in canonical histone incorporation patterns contribute to altered chromatin states in cancer.

How can HIST1H2AG (Ab-36) antibody be used to study the role of this histone in gene regulation?

The study of histone variants in gene regulation requires specific experimental approaches:

• ChIP-seq analysis:

  • Map genome-wide distribution of HIST1H2AG

  • Compare with active/repressive histone marks (H3K4me3, H3K27me3)

  • Correlate with transcription factor binding sites

• RNA-association studies:

  • Similar to studies with H2A.B, investigate whether HIST1H2AG associates with RNA processing factors

  • Analyze co-immunoprecipitation with RNA Polymerase II components

  • Perform RIP-seq to identify associated RNA species

• Nucleosome dynamics:

  • Use RhIP (recombinant histone immunoprecipitation) approaches to study incorporation dynamics

  • Analyze replication-dependent deposition compared to other H2A variants

  • Study interactions with histone chaperones like CAF-1 or HIRA

Research on H2A variants has shown distinct genomic localization patterns and functions. While H2A.B.3 shows elevated abundance at the beginning of gene bodies (in contrast to H3K36me3 enrichment at gene ends) , the specific distribution pattern of HIST1H2AG remains to be fully characterized.