HIST1H2AG (Ab-9) Antibody

What is HIST1H2AG (Ab-9) Antibody and what target does it recognize?

HIST1H2AG (Ab-9) is a polyclonal antibody generated in rabbits that specifically recognizes a peptide sequence around the lysine 9 (Lys9) site of Human Histone H2A type 1. This antibody belongs to the IgG isotype and is typically supplied in an unconjugated format. The target protein is a core histone that functions as a component of the nucleosome, which wraps and compacts DNA into chromatin, limiting DNA accessibility to the cellular machinery involved in processes like transcription and replication .

What are the technical specifications of HIST1H2AG (Ab-9) Antibody?

The HIST1H2AG (Ab-9) Antibody has the following specifications:

The antibody is produced using a synthetic peptide as immunogen, which contains the sequence surrounding lysine 9 of human histone H2A type 1 .

How does HIST1H2AG relate to other histone H2A variants and what are its synonyms?

HIST1H2AG is one of several histone H2A variants encoded in the human genome. The protein is also known by several synonyms including:

• H2AC11

• H2AFP

• H2AC13

• H2AFC

• HIST1H2AI

• H2AC15

• H2AFD

• HIST1H2AK

• H2AC16

• H2AFI

• HIST1H2AL

• H2AC17

• H2AFN

• HIST1H2AM

• Histone H2A type 1

• H2A.1

• Histone H2A/ptl

The UniProt accession number for this protein is P0C0S8 . Understanding these relationships is important for researchers to ensure they are targeting the correct histone variant in their experiments, as histone variants can have distinct functions in chromatin regulation.

What are the validated applications for HIST1H2AG (Ab-9) Antibody?

HIST1H2AG (Ab-9) Antibody has been validated for several common immunological techniques in research:

• ELISA (Enzyme-Linked Immunosorbent Assay): Useful for quantitative detection of the target protein in solution .

• Western Blot (WB): Enables detection of the target protein in cell or tissue lysates, allowing determination of protein size and semi-quantitative analysis .

• Immunohistochemistry (IHC): Facilitates visualization of the target protein in tissue sections, providing information about protein localization within tissues and cells .

• Immunofluorescence (IF): Allows for high-resolution imaging of protein localization at the subcellular level .

For optimal results in each application, researchers should follow specific protocols and optimize conditions including antibody dilution, incubation time, and detection method based on their experimental system.

How should HIST1H2AG (Ab-9) Antibody be used for Chromatin Immunoprecipitation (ChIP) experiments?

While the HIST1H2AG (Ab-9) Antibody specification sheet doesn't explicitly list ChIP as a validated application, many histone antibodies are suitable for ChIP experiments. Based on data from similar histone antibodies such as HIST1H2AG (Ab-95) , the following protocol outline is recommended:

• Cross-linking: Fix cells with 1% formaldehyde for 10 minutes at room temperature to preserve protein-DNA interactions.

• Chromatin preparation: Lyse cells and sonicate chromatin to fragments of 200-500 bp.

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate chromatin with 2-5 μg of HIST1H2AG antibody overnight at 4°C

  • Add protein A/G beads and incubate for 1-2 hours

  • Wash beads thoroughly to remove non-specific binding

• Reverse cross-linking and DNA purification: Elute DNA-protein complexes, reverse crosslinks, and purify DNA.

• Analysis: Analyze precipitated DNA by qPCR, sequencing, or other methods.

For accurate ChIP measurements, incorporation of spike-in chromatin as an internal control is recommended, similar to the approach used in H3K9 acetylation ChIP assays .

What are the recommended sample preparation methods for Western blot using HIST1H2AG (Ab-9) Antibody?

For optimal Western blot results with HIST1H2AG (Ab-9) Antibody, follow these methodological recommendations:

• Histone extraction: Due to histones' basic nature and tight association with DNA, specialized extraction protocols are recommended:

  • Use a histone extraction kit or an acid extraction method (e.g., 0.2N HCl)

  • Alternative method: Extract with high salt buffer followed by TCA precipitation

• Sample preparation:

  • Dissolve extracted histones in SDS sample buffer

  • Heat at 95°C for 5 minutes

  • Load 5-20 μg of histone extract per lane

• Gel electrophoresis:

  • Use 15-18% SDS-PAGE gels to properly resolve low molecular weight histones

  • Include a molecular weight marker that covers the 10-20 kDa range

• Transfer and detection:

  • Transfer to PVDF membrane (recommended over nitrocellulose for histone proteins)

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

  • Incubate with primary antibody at optimal dilution (typically 1:500-1:2000)

  • Use HRP-conjugated or fluorescently-labeled secondary antibodies for detection

The expected molecular weight of histone H2A is approximately 14 kDa. When interpreting results, consider potential post-translational modifications which may affect protein migration.

How can HIST1H2AG (Ab-9) Antibody be used to study histone modifications and their relation to gene expression?

HIST1H2AG (Ab-9) Antibody can be utilized in multi-faceted experimental approaches to study the relationship between histone H2A and its modifications in gene regulation:

• ChIP-seq integration: Combine ChIP using HIST1H2AG antibody with next-generation sequencing to map genome-wide distribution of H2A. This can be integrated with RNA-seq data to correlate H2A positioning with gene expression patterns, similar to studies done with H2A.Z isoforms .

• Sequential ChIP (Re-ChIP): To study co-occurrence of H2A with specific modifications:

  • Perform first ChIP with HIST1H2AG antibody

  • Elute complexes under non-denaturing conditions

  • Perform second ChIP with antibodies against histone modifications (e.g., H2AK119ub)

  • This approach can reveal how unmodified versus modified H2A correlates with gene activation or repression

• Comparative epigenetic profiling: Compare HIST1H2AG distribution with deubiquitinases like BAP1 that remove the repressive H2AK119ub mark, which has been shown to play crucial roles in B-cell development and humoral immune responses .

• Functional validation: Combine ChIP data with genetic approaches (CRISPR-Cas9) to modify specific regions where HIST1H2AG is enriched and analyze effects on gene expression and cell function.

When designing these experiments, it's crucial to include appropriate controls and to consider the dynamic nature of histone modifications in different cellular contexts.

What are the key considerations for analyzing potential cross-reactivity of HIST1H2AG (Ab-9) Antibody with other histone variants?

When analyzing potential cross-reactivity of HIST1H2AG (Ab-9) Antibody with other histone variants, researchers should consider:

• Sequence homology analysis:

  • H2A family proteins share high sequence similarity, particularly around conserved regions

  • The antibody targets the region around Lys9, which may have varying degrees of conservation among H2A variants

  • Perform sequence alignments of the immunogen peptide against all H2A variants to predict potential cross-reactivity

• Experimental validation strategies:

  • Peptide competition assays: Pre-incubate antibody with excess immunogen peptide before application; signal reduction confirms specificity

  • Knockout/knockdown controls: Use cells with CRISPR-mediated knockout or siRNA knockdown of HIST1H2AG as negative controls

  • Recombinant protein arrays: Test antibody against a panel of purified recombinant H2A variants to quantify cross-reactivity

• Mass spectrometry validation:

  • Perform immunoprecipitation with HIST1H2AG antibody

  • Analyze precipitated proteins by mass spectrometry

  • Identify all bound proteins to determine specificity for HIST1H2AG versus other histone variants

• Comparative antibody analysis:

  • Test multiple antibodies targeting different epitopes of HIST1H2AG

  • Compare binding patterns across cellular contexts and experimental conditions

Understanding cross-reactivity is particularly important when studying specialized histone variants like H2A.Z isoforms, which have distinct functions in gene regulation as demonstrated by integrated RNA-seq and chromatin studies .

How can epigenetic data obtained using HIST1H2AG (Ab-9) Antibody be integrated with other datasets to understand chromatin regulatory networks?

Integrating epigenetic data from HIST1H2AG (Ab-9) Antibody experiments with other datasets requires sophisticated bioinformatic approaches:

• Multi-omics data integration:

  • Combine ChIP-seq data for HIST1H2AG with:

    • RNA-seq to correlate with gene expression

    • ATAC-seq to associate with chromatin accessibility

    • Hi-C or similar techniques to link with 3D chromatin organization

    • Proteomics data to identify interacting protein complexes

  • This multi-dimensional approach provides a comprehensive view of how H2A contributes to chromatin regulation

• Advanced computational analysis:

  • Apply machine learning algorithms to identify patterns in integrated datasets

  • Use network analysis to construct gene regulatory networks with H2A as a component

  • Implement statistical methods like multivariate analysis to identify significant correlations between H2A localization and other epigenetic features

• Functional validation approaches:

  • Design perturbation experiments targeting regions with significant HIST1H2AG enrichment

  • Use CRISPR-based techniques for precise epigenetic editing

  • Compare results with predefined models from integrated data analysis

• Temporal and context-specific analysis:

  • Generate time-course data to understand dynamic changes in H2A localization

  • Compare datasets across different cell types or treatment conditions

  • Analyze context-specific functions, similar to the B-cell specific studies of BAP1 and H2AK119ub

This integrated approach can reveal how HIST1H2AG contributes to complex epigenetic regulation, similar to studies that have revealed antagonistic functions of histone modifiers like PHF14 and SIRT1 in regulating H3K9 acetylation at specific promoters .

What are common troubleshooting strategies for weak or non-specific signals when using HIST1H2AG (Ab-9) Antibody?

When encountering weak or non-specific signals with HIST1H2AG (Ab-9) Antibody, consider these methodological troubleshooting approaches:

• For weak signals:

  • Antibody concentration: Increase antibody concentration (reduce dilution)

  • Incubation time: Extend primary antibody incubation (overnight at 4°C)

  • Detection enhancement: Use signal amplification systems (e.g., biotin-streptavidin)

  • Sample preparation: Optimize histone extraction protocol to improve yield

  • Epitope retrieval: For IHC/IF, test different antigen retrieval methods (citrate, EDTA buffers at various pH)

  • Blocking agents: Test alternative blocking reagents (BSA vs. normal serum vs. commercial blockers)

• For non-specific signals:

  • Antibody dilution: Increase dilution to reduce non-specific binding

  • Blocking optimization: Increase blocking reagent concentration or time

  • Wash conditions: Increase stringency of wash steps (more washes, higher salt)

  • Secondary antibody: Test alternative secondary antibodies or pre-adsorbed versions

  • Tissue fixation: For IHC/IF, optimize fixation protocol (duration, fixative type)

  • Negative controls: Include no-primary-antibody controls and isotype controls

• Application-specific considerations:

  • Western blot: Use gradient gels to better resolve histone proteins

  • ChIP: Optimize sonication conditions for consistent chromatin fragmentation

  • IHC/IF: Test different fixation and permeabilization methods to improve epitope accessibility

• Sample-specific considerations:

  • Protein abundance: Consider sample enrichment techniques for low-abundance targets

  • Post-translational modifications: Be aware that modifications near the epitope may affect antibody binding

  • Sample age/integrity: Use fresh samples or properly stored samples to preserve epitopes

Careful optimization of these parameters can significantly improve experimental outcomes with HIST1H2AG (Ab-9) Antibody.

How can HIST1H2AG (Ab-9) Antibody be effectively used in multiplex immunofluorescence studies with other histone modification antibodies?

Multiplexed immunofluorescence with HIST1H2AG (Ab-9) Antibody and other histone modification antibodies requires careful planning:

• Antibody selection and compatibility:

  • Choose antibodies raised in different host species to avoid cross-reactivity

  • For co-detection with HIST1H2AG (rabbit polyclonal), select companion antibodies raised in mouse, rat, or goat

  • Verify that epitopes are accessible simultaneously (some fixation methods may preferentially preserve certain epitopes)

• Experimental design strategies:

  • Sequential staining protocol:

    1. Apply first primary antibody (e.g., HIST1H2AG)

    2. Detect with fluorophore-conjugated secondary antibody

    3. Block remaining binding sites

    4. Apply second primary antibody (e.g., H3K9ac antibody)

    5. Detect with different fluorophore-conjugated secondary antibody

  • Directly conjugated antibodies:

    • Consider using directly conjugated versions of antibodies to eliminate secondary antibody cross-reactivity

    • Custom conjugation services are available for HIST1H2AG antibody

• Controls for multiplex experiments:

  • Single-antibody controls to establish baseline signals

  • Secondary-only controls to check for non-specific binding

  • Peptide competition controls to verify specificity

  • Fluorophore spectral controls to check for bleed-through

• Advanced imaging considerations:

  • Use spectral imaging and linear unmixing for closely emitting fluorophores

  • Implement sequential scanning for confocal microscopy

  • Consider super-resolution techniques for detailed co-localization studies

  • Use appropriate image analysis software for quantitative co-localization measurements

This approach enables researchers to visualize and quantify the spatial relationships between HIST1H2AG and other histone marks, similar to studies examining relationships between histone modifications like H3K9 acetylation and other epigenetic markers .

What strategies can be employed for optimizing HIST1H2AG (Ab-9) Antibody performance in challenging tissue samples?

When working with challenging tissue samples, optimize HIST1H2AG (Ab-9) Antibody performance using these methodological approaches:

• Tissue fixation and processing optimization:

  • Fixation timing: Minimize over-fixation which can mask epitopes (limit to 24-48 hours in formalin)

  • Fixative selection: Compare different fixatives (formalin, paraformaldehyde, alcohol-based)

  • Processing parameters: Optimize dehydration and clearing steps to preserve tissue architecture

  • Section thickness: Test different section thicknesses (4-7 μm for FFPE, 10-20 μm for frozen)

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER):

    • Test multiple buffer systems (citrate pH 6.0, EDTA pH 8.0, Tris-EDTA pH 9.0)

    • Compare different heating methods (microwave, pressure cooker, water bath)

    • Optimize heating times (10-30 minutes)

  • Enzymatic retrieval:

    • Test protease-based methods (proteinase K, trypsin)

    • Optimize enzyme concentration and incubation time

  • Combination approaches:

    • Sequential application of HIER followed by mild enzymatic treatment

• Signal enhancement strategies:

  • Amplification systems:

    • Tyramide signal amplification (TSA)

    • Polymer-based detection systems

    • Nanobody-based detection

  • Background reduction:

    • Dual endogenous enzyme blocking (peroxidase and alkaline phosphatase)

    • Avidin/biotin blocking for high biotin samples

    • Sudan Black B treatment for tissues with high autofluorescence

• Special considerations for difficult tissues:

  • Highly fibrotic tissues: Extended antigen retrieval and/or partial digestion with collagenase

  • Fatty tissues: Additional deparaffinization steps or detergent treatments

  • Necrotic tissues: Careful region selection and modified blocking procedures

  • Archival materials: Adjusted retrieval times and enhanced detection methods

These methodological optimizations can significantly improve the detection of HIST1H2AG in challenging samples, enabling more consistent and reliable research results.

How can HIST1H2AG (Ab-9) Antibody be used to investigate the relationship between histone H2A and epigenetic regulators like BAP1?

HIST1H2AG (Ab-9) Antibody can be used in sophisticated experimental designs to explore interactions between histone H2A and epigenetic regulators like the deubiquitinase BAP1:

• Sequential ChIP (Re-ChIP) protocol:

  • First ChIP: Immunoprecipitate with HIST1H2AG antibody

  • Second ChIP: Re-immunoprecipitate with BAP1 antibody

  • Analyze regions where both proteins co-occupy chromatin

  • This reveals direct interactions between BAP1 and H2A at specific genomic loci

• Comparative ChIP-seq analysis:

  • Perform parallel ChIP-seq with HIST1H2AG antibody and BAP1 antibody

  • Compare genome-wide distribution patterns

  • Identify regions of overlap and mutual exclusion

  • Correlate with H2AK119ub levels and gene expression data

  • This approach can reveal how BAP1 regulates the genome-wide H2AK119ub landscape, similar to studies in B-cell activation

• Functional studies using genetic manipulation:

  • Create BAP1 knockout or knockdown cell lines

  • Analyze changes in HIST1H2AG distribution and H2AK119ub levels

  • Perform rescue experiments with wild-type vs. catalytically inactive BAP1

  • This strategy can determine how BAP1's deubiquitinase activity affects H2A distribution and function

• Protein-protein interaction studies:

  • Immunoprecipitate with HIST1H2AG antibody and probe for BAP1

  • Perform proximity ligation assays to visualize in situ interactions

  • Use mass spectrometry to identify all proteins in H2A-containing complexes

  • These approaches can identify direct and indirect interactions between H2A and BAP1

Understanding these relationships has significant implications for comprehending epigenetic regulation in B-cell development, immune responses, and related pathologies as demonstrated in recent studies .

What are the methodological considerations for using HIST1H2AG (Ab-9) Antibody in studying chromatin dynamics during cell differentiation?

Studying chromatin dynamics during cell differentiation with HIST1H2AG (Ab-9) Antibody requires careful methodological planning:

• Temporal experimental design:

  • Time-course sampling: Collect cells at defined stages of differentiation

  • Synchronization strategies: Use methods to synchronize cell populations

  • Single-cell approaches: Consider single-cell techniques to address population heterogeneity

  • Live-cell imaging: For dynamic studies, consider using tagged histones in parallel with fixed-cell antibody approaches

• Integration with differentiation markers:

  • Co-staining protocols:

    • Combine HIST1H2AG antibody with lineage-specific markers

    • Use different fluorophores with minimal spectral overlap

    • Apply sequential staining if working with challenging antibody combinations

  • Flow cytometry integration:

    • Develop protocols for intracellular HIST1H2AG staining compatible with surface marker detection

    • Use sorting to isolate specific subpopulations for detailed analysis

• ChIP-seq methodological adaptations:

  • Low-cell-number protocols: Optimize for limited cell numbers at specific differentiation stages

  • Normalization strategies: Use spike-in controls for accurate quantitative comparisons between time points

  • Bioinformatic analysis: Implement trajectory analysis to track chromatin changes over differentiation time

• Functional validation approaches:

  • Targeted epigenetic editing: Use CRISPR-Cas9-based approaches to modify histones at specific loci

  • Differentiation assays: Assess how perturbation of H2A or its modifiers affects differentiation outcomes

  • Rescue experiments: Restore wild-type histones in modified cells to confirm specificity

These methodological approaches can help researchers uncover how HIST1H2AG distribution and modifications change during differentiation processes, similar to studies examining epigenetic regulation in B-cell development and activation .

How can HIST1H2AG (Ab-9) Antibody be used in combination with genome editing techniques to study histone function?

Combining HIST1H2AG (Ab-9) Antibody with genome editing techniques offers powerful approaches to study histone function:

• CRISPR-Cas9-based strategies for histone modification:

  • Tag endogenous HIST1H2AG:

    • Insert epitope tags (FLAG, HA) at the endogenous locus

    • Use homology-directed repair (HDR) with donor templates

    • Validate edited cells using the HIST1H2AG antibody as a control

    • This approach was successfully used for PHF14 CRISPR editing

  • Create specific histone mutations:

    • Generate point mutations at key residues (e.g., modification sites)

    • Use the HIST1H2AG antibody to assess effects on chromatin distribution

    • Similar approaches have been used for H2AFZ editing by homology directed repair

  • Conditional knockout systems:

    • Implement inducible CRISPR systems for temporal control

    • Use HIST1H2AG antibody to confirm depletion and study consequences

• Methodological workflow for genome editing validation:

  • Design phase:

    • Select appropriate guide RNAs using tools like CRISPOR

    • Design HDR templates with desired modifications

    • Include selection markers or screening strategies

  • Delivery methods:

    • Optimize transfection for cell type (electroporation, lipofection)

    • Consider viral delivery for difficult-to-transfect cells

    • Use co-selection strategies (e.g., ouabaine-based selection)

  • Validation protocol:

    • PCR screening of edited clones

    • Sequencing confirmation

    • Western blot with HIST1H2AG antibody

    • ChIP-qPCR to assess chromatin distribution

• Advanced applications:

  • CUT&RUN or CUT&Tag with HIST1H2AG antibody:

    • Apply in edited cells to map genome-wide distribution changes

    • Compare wild-type versus mutant histones

  • Epigenetic editing:

    • Target specific loci with dCas9 fused to histone modifiers

    • Use HIST1H2AG antibody to assess effects on recruitment and spreading

  • Synthetic genetic interactions:

    • Combine histone mutations with modifications to readers, writers, or erasers

    • Study compensatory mechanisms in chromatin regulation

These approaches enable precise dissection of histone function in various cellular contexts, providing mechanistic insights into chromatin regulation.

What are emerging applications for HIST1H2AG (Ab-9) Antibody in cutting-edge epigenetic research?

HIST1H2AG (Ab-9) Antibody has potential applications in several frontier areas of epigenetic research:

• Single-cell epigenomics:

  • Integration with single-cell sequencing technologies

  • Development of micro-ChIP protocols using HIST1H2AG antibody

  • Spatial profiling of histone variants in complex tissues

  • These approaches can reveal cell-to-cell heterogeneity in histone distribution and modification

• Multi-omics integration:

  • Combined analysis of histone positioning, DNA methylation, and gene expression

  • Correlation with chromatin accessibility and 3D genome organization

  • Integration with proteomics to identify context-specific protein interactions

  • These integrative approaches provide comprehensive views of epigenetic regulation

• Liquid biopsy applications:

  • Detection of circulating nucleosomes containing HIST1H2AG

  • Analysis of histone modifications in cell-free DNA-protein complexes

  • Potential biomarker development for diseases with epigenetic dysregulation

• Therapeutic targeting studies:

  • Screening for compounds that modulate histone variant incorporation

  • Monitoring effects of epigenetic drugs on histone distribution

  • Developing targeted approaches to modify specific histone variants

These emerging applications position HIST1H2AG (Ab-9) Antibody as a valuable tool for researchers exploring the frontiers of epigenetic regulation in health and disease contexts.

How might methodological advances improve the utility of HIST1H2AG (Ab-9) Antibody in future research?

Future methodological advances could significantly enhance the utility of HIST1H2AG (Ab-9) Antibody:

• Technical improvements in antibody development:

  • Generation of monoclonal versions for increased specificity

  • Development of recombinant antibodies with defined binding characteristics

  • Creation of antibody fragments (Fab, scFv) for improved tissue penetration

  • These advances could increase specificity and reproducibility across experiments

• Advanced imaging applications:

  • Super-resolution microscopy optimization for histone variant visualization

  • Live-cell imaging compatible antibody derivatives

  • Expansion microscopy protocols for improved spatial resolution

  • These techniques would enable detailed visualization of histone dynamics in living cells

• High-throughput adaptations:

  • Microfluidic ChIP systems for automated processing

  • Barcoded antibody approaches for multiplexed detection

  • Integration with single-cell platforms for population analysis

  • These developments would increase experimental throughput and data generation

• Computational integration:

  • Machine learning algorithms to interpret complex histone patterns

  • Predictive modeling of histone variant function based on distribution

  • Systems biology approaches to integrate histone data with other cellular parameters

  • These computational tools would enhance data interpretation and hypothesis generation

These methodological advances would expand the research applications of HIST1H2AG (Ab-9) Antibody and provide deeper insights into histone biology and epigenetic regulation.

What is the significance of studying histone variants like HIST1H2AG in understanding disease mechanisms?

Studying histone variants like HIST1H2AG has profound implications for understanding disease mechanisms:

• Cancer biology connections:

  • Altered histone variant incorporation is associated with various cancers

  • Epigenetic regulators like BAP1 that interact with histones function as tumor suppressors

  • HIST1H2AG distribution patterns may serve as prognostic biomarkers

  • Understanding these relationships could lead to novel diagnostic and therapeutic approaches

• Immune system regulation:

  • Histone variants play crucial roles in immune cell development and function

  • BAP1-mediated deubiquitination of H2AK119ub affects B-cell activation and antibody production

  • Dysregulation of histone modifications can contribute to autoimmune conditions

  • These insights could inform development of immunomodulatory therapies

• Developmental disorders:

  • Proper histone variant incorporation is essential for normal development

  • Mutations in histone genes or their regulators can cause developmental abnormalities

  • Histone modification patterns establish and maintain cell identity

  • Understanding these processes may lead to interventions for developmental disorders

• Aging and neurodegeneration:

  • Chromatin structure changes with age and in neurodegenerative diseases

  • Histone variant distribution affects genome stability and DNA repair

  • Epigenetic interventions targeting histone biology show promise for age-related conditions

  • These connections highlight the importance of histone research for healthy aging