HIST1H2AG (Ab-118) Antibody

What is HIST1H2AG (Ab-118) Antibody and what does it target?

HIST1H2AG (Ab-118) is a rabbit polyclonal antibody that specifically targets Human Histone H2A type 1, a core component of nucleosomes. The antibody recognizes a peptide sequence surrounding lysine 118 of human histone H2A type 1 (Uniprot Accession: P0C0S8). This antibody is instrumental in studying chromatin structure, histone modifications, and epigenetic regulation mechanisms. Histones play critical roles in transcription regulation, DNA repair, DNA replication, and chromosomal stability, making this antibody valuable for epigenetic research .

What species reactivity has been validated for this antibody?

The HIST1H2AG (Ab-118) polyclonal antibody has been validated for reactivity with human (Homo sapiens) samples, as consistently reported across sources. Some suppliers also indicate cross-reactivity with mouse (Mus musculus) samples . When working with species not explicitly listed, preliminary validation experiments are strongly recommended to confirm specificity before proceeding with full-scale experiments .

What applications has HIST1H2AG (Ab-118) Antibody been validated for?

This antibody has been validated for multiple applications including:

• ELISA (Enzyme-Linked Immunosorbent Assay)

• IHC (Immunohistochemistry)

• IF (Immunofluorescence)

• ChIP (Chromatin Immunoprecipitation)

• WB (Western Blotting)

• IP (Immunoprecipitation)

Recommended dilutions vary by application: WB (1:100-1:1000), IP (1:200-1:2000), IHC (1:10-1:100), and IF (1:1-1:10). These ranges should be optimized for specific experimental conditions and sample types .

How should I design a ChIP experiment using HIST1H2AG (Ab-118) Antibody?

For optimal ChIP experiments with HIST1H2AG (Ab-118) Antibody:

• Cell Preparation: Culture approximately 10^6 cells (as validated with HeLa cells).

• Chromatin Preparation: Treat cells with Micrococcal Nuclease to fragment chromatin to appropriate sizes (200-500 bp).

• Sonication: Apply gentle sonication to further fragment and solubilize chromatin.

• Immunoprecipitation: Use 5 μg of HIST1H2AG antibody per reaction. Always include a control reaction with normal rabbit IgG.

• DNA Recovery: Purify DNA using standard protocols after reverse crosslinking.

• Analysis: Quantify enrichment using real-time PCR with appropriate primers, such as those against the β-Globin promoter as previously validated .

The antibody has been shown to effectively immunoprecipitate histone H2A in ChIP applications, allowing for analysis of histone distribution and modifications across genomic regions .

What are the recommended protocols for Western blot analysis using this antibody?

For Western blot analysis using HIST1H2AG (Ab-118) Antibody:

• Sample Preparation: Prepare whole cell lysates (validated with NIH/3T3, A549, K562, and HepG2 cells).

• Protein Separation: Use appropriate percentage gels suitable for low molecular weight proteins (15-18% acrylamide gels recommended).

• Transfer: Optimize transfer conditions for small proteins (wet transfer recommended).

• Blocking: Block membranes using standard protocols (5% non-fat dry milk or BSA).

• Primary Antibody: Dilute HIST1H2AG antibody at 1:100-1:1000 (optimal concentration determined as 1 μg/ml in validation studies).

• Secondary Antibody: Use goat anti-rabbit IgG-HRP at 1:50000 dilution.

• Detection: Employ standard chemiluminescence detection methods.

The expected band size is 15 kDa, which corresponds to the predicted molecular weight of histone H2A .

What are common issues when using HIST1H2AG antibody in immunofluorescence and how can they be resolved?

Common challenges in immunofluorescence experiments with HIST1H2AG antibody include:

• High Background Signal:

  • Solution: Increase blocking time (2 hours at room temperature)

  • Optimize antibody dilution (start with 1:5 dilution and titrate as needed)

  • Include additional washing steps (at least 3×10 minutes in PBS-T)

  • Use specialized blocking agents containing both serum and BSA

• Weak or No Signal:

  • Solution: Optimize fixation method (try 4% paraformaldehyde for 15 minutes)

  • Include an antigen retrieval step if using fixed tissues

  • Increase antibody concentration or incubation time (overnight at 4°C recommended)

  • Enhance detection with signal amplification systems

• Non-specific Nuclear Staining:

  • Solution: Pre-adsorb antibody with acetone powder from relevant cell types

  • Include additional blocking steps specific for nuclear proteins

  • Optimize permeabilization conditions (0.1% Triton X-100 for 10 minutes)

How can I optimize immunoprecipitation protocols with HIST1H2AG (Ab-118) Antibody?

To optimize immunoprecipitation with HIST1H2AG antibody:

• Lysate Preparation:

  • Use fresh cell lysates (500 μg total protein recommended)

  • Include protease and phosphatase inhibitors

  • Optimize lysis buffer components (test both RIPA and gentler NP-40 based buffers)

• Antibody Amount:

  • Start with 5 μg antibody per 500 μg protein lysate

  • Pre-clear lysate with Protein G beads before adding antibody

• Incubation Conditions:

  • Extend antibody-lysate incubation to overnight at 4°C with gentle rotation

  • Use protein G-conjugated magnetic beads for cleaner precipitations

• Washing Stringency:

  • Include graduated washing steps with decreasing salt concentrations

  • Use at least 4-5 wash steps to reduce background

• Elution Method:

  • Compare direct elution in SDS loading buffer vs. mild elution for functional studies

  • For challenging IPs, consider crosslinking the antibody to beads

These optimizations have been validated using NIH/3T3 whole cell lysates, where specific immunoprecipitation of HIST1H2AG was confirmed by Western blotting .

How can HIST1H2AG (Ab-118) Antibody be used to study histone post-translational modifications?

HIST1H2AG antibody can be instrumental in studying histone post-translational modifications through several approaches:

• Sequential ChIP (ChIP-reChIP):

  • First immunoprecipitate with HIST1H2AG antibody

  • Elute under mild conditions

  • Perform second ChIP with antibodies against specific modifications (e.g., acetylation, methylation)

  • This reveals which modifications co-occur with specific H2A variants

• Mass Spectrometry Coupling:

  • Immunoprecipitate nucleosomes using HIST1H2AG antibody

  • Analyze by mass spectrometry to identify post-translational modifications

  • This approach provides comprehensive modification landscapes

• Comparative ChIP-seq Analysis:

  • Perform parallel ChIP-seq with HIST1H2AG antibody and modification-specific antibodies

  • Compare genomic distribution patterns

  • Identify regions enriched for both the histone variant and specific modifications

Since histones undergo complex post-translational modifications that regulate DNA accessibility and transcription, this antibody serves as an important tool for understanding the interplay between histone variants and their modification patterns in epigenetic regulation .

What considerations are important when using HIST1H2AG antibody in tumor tissue samples?

When examining tumor tissues with HIST1H2AG antibody, researchers should consider:

• Tissue Fixation and Processing:

  • Formalin fixation time critically affects histone epitope accessibility

  • Recommend 24-hour fixation in 10% neutral buffered formalin

  • Use antigen retrieval methods (heat-induced epitope retrieval in citrate buffer pH 6.0)

• Internal Controls:

  • Always include normal tissue sections as controls

  • Utilize known positive cell lines (HepG2, K562) as reference standards

  • Consider dual immunostaining with other nuclear markers for context

• Tumor Heterogeneity Considerations:

  • Analyze multiple tumor regions due to potential epigenetic heterogeneity

  • Consider tumor grade/stage-specific variations in histone patterns

  • Record and analyze spatial distribution patterns of staining

• Interpretation Challenges:

  • Account for variations in histone modifications between tumor types

  • Distinguish between changes in histone variant expression versus modifications

  • Correlate findings with other epigenetic markers

How does HIST1H2AG function within the broader context of chromatin biology?

HIST1H2AG encodes a member of the histone H2A family, which is one of the core components of the nucleosome. Its biological significance includes:

• Nucleosome Structure: H2A proteins pair with H2B to form heterodimers that are essential for nucleosome assembly. Each nucleosome contains two H2A-H2B dimers along with an H3-H4 tetramer, around which approximately 147 bp of DNA wraps.

• Chromatin Regulation: H2A variants influence chromatin compaction states, affecting DNA accessibility to transcription machinery and other DNA-interacting proteins.

• Transcriptional Control: Distribution of H2A variants correlates with transcriptional activity, with certain regions of the genome showing enrichment or depletion of specific variants.

• Cell Cycle Regulation: Expression of histone H2A variants is tightly regulated during the cell cycle, particularly during S phase when DNA replication occurs.

• Interaction with Chromatin Remodeling Complexes: H2A histones interact with various chromatin remodeling complexes that regulate nucleosome positioning and modification .

How can I validate the specificity of HIST1H2AG (Ab-118) Antibody in my experimental system?

To validate antibody specificity in your experimental system:

• Peptide Competition Assay:

  • Pre-incubate the antibody with the immunizing peptide (sequence around Lys-118)

  • Run parallel experiments with blocked and unblocked antibody

  • Signal disappearance with blocked antibody confirms specificity

• Knockout/Knockdown Controls:

  • Use cell lines with CRISPR/Cas9 knockout or siRNA knockdown of HIST1H2AG

  • Compare signal between wild-type and knockout/knockdown samples

  • Signal reduction in knockdown samples validates specificity

• Multiple Antibody Validation:

  • Test multiple antibodies against different epitopes of HIST1H2AG

  • Compare staining/binding patterns

  • Consistent results across antibodies increase confidence in specificity

• Mass Spectrometry Confirmation:

  • Immunoprecipitate with HIST1H2AG antibody

  • Analyze pulled-down proteins by mass spectrometry

  • Confirm predominant recovery of HIST1H2AG and associated proteins

• Cross-Reactivity Testing:

  • Test the antibody against recombinant proteins of different H2A variants

  • Quantify relative binding affinities

  • Ensure minimal cross-reactivity with other H2A family members

What are the key differences in ChIP-seq vs. ChIP-qPCR protocols when using HIST1H2AG antibody?

When using HIST1H2AG antibody for chromatin studies, the differences between ChIP-seq and ChIP-qPCR approaches include:

ChIP-seq Protocol Adaptations:

• Input Material: Require larger starting material (typically 10^7 cells vs. 10^6 for ChIP-qPCR)

• Chromatin Fragmentation: More stringent size selection (150-300 bp optimal for sequencing)

• IP Conditions: Higher antibody amounts (10 μg recommended vs. 5 μg for qPCR)

• Library Preparation: Additional steps for end repair, adapter ligation, and amplification

• Controls: Include input DNA, IgG control, and spike-in normalization standards

ChIP-qPCR Specific Considerations:

• Primer Design: Critical for specific amplification of target regions

• Controls: Include positive control regions (β-Globin promoter validated)

• Quantification: Calculate percent input or fold enrichment over IgG

• Replication: Minimum of three biological replicates recommended

Data Analysis Differences:

• ChIP-seq: Genome-wide analysis requiring bioinformatics pipeline

• ChIP-qPCR: Focused analysis of specific loci with simple statistical tests

Both approaches have been validated with the HIST1H2AG antibody, with ChIP-qPCR being particularly useful for targeted validation of specific genomic regions of interest .

How can I design experiments to study HIST1H2AG dynamics during cell cycle progression?

To study HIST1H2AG dynamics across the cell cycle:

• Cell Synchronization Approach:

  • Synchronize cells using double thymidine block (G1/S boundary)

  • Alternative: nocodazole treatment (M phase)

  • Collect cells at defined time points after release

  • Perform Western blot, immunofluorescence, or ChIP with HIST1H2AG antibody

• Flow Cytometry Integration:

  • Fix cells and stain with HIST1H2AG antibody

  • Co-stain with propidium iodide or DAPI for DNA content

  • Sort cells based on cell cycle phases

  • Quantify HIST1H2AG signal intensity per cell cycle phase

• Live-Cell Imaging Setup:

  • Create fluorescent protein-tagged H2A constructs

  • Validate co-localization with HIST1H2AG antibody staining

  • Perform time-lapse microscopy during cell division

  • Quantify protein dynamics in real-time

• ChIP-seq Across Cell Cycle:

  • Perform ChIP-seq with HIST1H2AG antibody in synchronized populations

  • Map changes in genomic distribution throughout cell cycle

  • Correlate with transcriptional changes and replication timing

Each approach provides complementary information about histone dynamics, with special consideration for potential epitope masking during mitotic chromatin condensation .

How does HIST1H2AG compare to other H2A variants in experimental applications?

Comparative analysis of HIST1H2AG versus other H2A variants reveals important experimental considerations:

When designing multi-parameter studies:

• Sequential Immunoprecipitation: Start with HIST1H2AG antibody followed by variant-specific antibodies

• Multiplexed Imaging: Combine HIST1H2AG with other variant antibodies raised in different host species

• Differential Extraction: Utilize salt fractionation to separate chromatin pools enriched for different variants

The choice between studying HIST1H2AG or other variants should be guided by the specific biological question, as each variant has distinct roles in chromatin regulation .

What are the cutting-edge research applications using HIST1H2AG antibody in cancer epigenetics?

Emerging applications of HIST1H2AG antibody in cancer epigenetics research include:

• Liquid Biopsy Development:

  • Detection of circulating nucleosomes containing HIST1H2AG

  • Correlation with tumor burden and treatment response

  • Integration with cell-free DNA sequencing data

• Drug Response Prediction:

  • ChIP-seq before and after epigenetic drug treatment

  • Identification of regions with altered HIST1H2AG occupancy

  • Correlation with transcriptional changes and clinical outcomes

• Tumor Heterogeneity Mapping:

  • Single-cell ChIP-seq or CUT&Tag with HIST1H2AG antibody

  • Spatial resolution of histone variant distribution in tumor sections

  • Integration with other omics data for comprehensive tumor profiling

• Therapeutic Target Identification:

  • Screening for proteins that specifically interact with HIST1H2AG

  • Identification of cancer-specific interactions

  • Development of targeted therapies disrupting these interactions

These advanced applications leverage the specificity of the HIST1H2AG antibody to understand fundamental epigenetic mechanisms in cancer development and progression .

What storage and handling practices maximize the performance and longevity of HIST1H2AG antibody?

To maintain optimal performance of HIST1H2AG (Ab-118) antibody:

• Storage Conditions:

  • Store at -20°C or -80°C for long-term stability

  • Avoid repeated freeze-thaw cycles (maximum 5 cycles recommended)

  • Aliquot upon first thaw to minimize freeze-thaw damage

  • Keep on ice when in use

• Buffer Considerations:

  • The antibody is supplied in buffer containing 50% glycerol, 0.01M PBS, pH 7.4, with 0.03% Proclin 300 as preservative

  • This formulation ensures stability during proper storage

  • Do not dilute the stock antibody until immediately before use

• Working Solution Preparation:

  • Prepare working dilutions fresh on the day of experiment

  • Use high-quality diluents (e.g., PBS with 1-5% BSA or normal serum)

  • Centrifuge briefly before opening to collect liquid at the bottom of the vial

  • Use sterile technique when handling to prevent contamination

• Transportation:

  • Ship on dry ice for extended transit

  • Monitor temperature during shipping

  • Allow to equilibrate to room temperature before opening

• Quality Control:

  • Test antibody performance periodically against known positive controls

  • Document lot numbers and correlate with experimental results

  • Consider validation with each new experimental system

How do I troubleshoot non-specific bands in Western blot using HIST1H2AG antibody?

When encountering non-specific bands in Western blot analysis with HIST1H2AG antibody:

• Sample Preparation Optimization:

  • Include protease inhibitors in lysis buffers

  • Maintain low temperature during extraction

  • Consider specialized histone extraction protocols

  • Test both denaturing and non-denaturing conditions

• Blocking Optimization:

  • Increase blocking time to 2 hours at room temperature

  • Test alternative blocking agents (5% milk vs. 3-5% BSA)

  • Add 0.1% Tween-20 to all buffers to reduce non-specific binding

• Antibody Dilution Strategy:

  • Create a dilution series (1:100, 1:500, 1:1000)

  • Extend primary antibody incubation to overnight at 4°C

  • Wash more extensively after antibody incubation (5-6 washes)

• Gel Separation Techniques:

  • Use higher percentage gels (15-18%) for better resolution of small proteins

  • Run the gel at lower voltage for improved separation

  • Consider using specialized gradient gels

• Positive Controls:

  • Include validated cell lines (NIH/3T3, A549, K562, or HepG2)

  • Compare with recombinant HIST1H2AG protein

  • Look for the expected 15 kDa band

The antibody has been validated to detect a specific 15 kDa band corresponding to histone H2A in multiple cell lines, with minimal non-specific binding when optimal conditions are used .

How can HIST1H2AG antibody be adapted for use in single-cell epigenomic profiling?

Adapting HIST1H2AG antibody for single-cell epigenomic applications:

• Single-Cell CUT&Tag Protocol:

  • Bind cells to concanavalin A-coated magnetic beads

  • Permeabilize cell membranes with digitonin

  • Incubate with HIST1H2AG antibody (1:50 dilution)

  • Add pA-Tn5 transposase to antibody

  • Tagmentation and library preparation from individual cells

• Epitope-Specific Antibody Optimization:

  • Test multiple concentrations (1:10 to 1:100)

  • Evaluate different permeabilization conditions

  • Validate with spike-in controls

  • Confirm specificity via comparison with bulk profiles

• Microfluidic Integration:

  • Encapsulate individual cells in droplets

  • Perform immunostaining with HIST1H2AG antibody

  • Analyze via droplet-based microfluidics

  • Combine with RNA-seq for multi-omic profiling

• Data Analysis Considerations:

  • Apply specialized bioinformatics pipelines for sparse data

  • Implement batch correction algorithms

  • Use dimensionality reduction techniques tailored to epigenomic data

  • Integrate with single-cell transcriptomics and genomics

These single-cell approaches reveal cell-to-cell variation in histone variant distribution that is masked in bulk assays, providing crucial information about epigenetic heterogeneity .

What are the considerations for using HIST1H2AG antibody in multiplexed imaging studies?

For multiplexed imaging with HIST1H2AG antibody:

• Antibody Compatibility Assessment:

  • Test for cross-reactivity with other primary antibodies

  • Select antibodies raised in different host species

  • Validate secondary antibody specificity to avoid cross-reaction

  • Consider direct fluorophore conjugation for challenging combinations

• Sequential Immunostaining Approach:

  • Perform initial staining with HIST1H2AG antibody

  • Image and record coordinates

  • Strip antibodies using glycine-SDS or commercial stripping buffers

  • Re-stain with next antibody set

  • Align images using fiducial markers

• Spectral Unmixing Requirements:

  • Use spectrally distinct fluorophores

  • Include single-stain controls for each fluorophore

  • Apply computational algorithms for unmixing

  • Consider autofluorescence removal strategies

• Optimized Nuclear Staining Protocol:

  • Use mild fixation (2% PFA for 10-15 minutes)

  • Perform antigen retrieval (heat-mediated in citrate buffer)

  • Extend permeabilization time (0.3% Triton X-100 for 15-20 minutes)

  • Use nuclear counterstains compatible with multiple imaging rounds

• Image Analysis Adaptations:

  • Implement nuclear segmentation algorithms

  • Quantify co-localization with other nuclear markers

  • Perform spatial distribution analysis

  • Correlate with functional genomic data

Multiplexed imaging reveals spatial relationships between HIST1H2AG and other nuclear proteins that cannot be detected by biochemical methods alone .

How can HIST1H2AG antibody data be integrated with other epigenomic datasets?

Integration of HIST1H2AG antibody-generated data with broader epigenomic datasets:

• Multi-omics Data Integration Framework:

  • Align ChIP-seq data with RNA-seq, ATAC-seq, and DNA methylation maps

  • Apply machine learning algorithms to identify correlative patterns

  • Generate integrated epigenomic profiles across genomic regions

  • Validate with orthogonal experimental approaches

• Public Database Comparison:

  • Compare HIST1H2AG binding patterns with ENCODE and Roadmap Epigenomics datasets

  • Identify cell-type specific and conserved binding patterns

  • Correlate with genomic annotations and functional elements

  • Develop prediction models for histone variant distribution

• Pathway Analysis Enhancement:

  • Map HIST1H2AG enrichment to genes in specific pathways

  • Correlate with transcriptional activity of pathway components

  • Identify potential epigenetic regulators of key cellular processes

  • Connect histone variant distribution to phenotypic outcomes

• Visualization Tools:

  • Develop genome browsers with integrated HIST1H2AG ChIP-seq tracks

  • Create interactive heatmaps for multi-sample comparisons

  • Implement circular visualization for long-range interactions

  • Generate 3D chromatin conformation models with HIST1H2AG mapping

This integrative approach reveals functional relationships between histone variant distribution and other epigenetic marks, providing deeper insights into chromatin regulation mechanisms .

What emerging technologies might enhance HIST1H2AG antibody applications in the future?

Emerging technologies poised to enhance HIST1H2AG antibody applications include:

• CUT&Tag Adaptations:

  • Integrates HIST1H2AG antibody with Tn5 transposase for direct chromatin tagmentation

  • Requires significantly less input material than traditional ChIP

  • Provides higher signal-to-noise ratio and resolution

  • Enables single-cell applications

• Proximity Ligation Developments:

  • Combines HIST1H2AG antibody with antibodies against histone modifications

  • Detects co-occurrence of modifications on the same nucleosomes

  • Reveals combinatorial histone codes at single-molecule resolution

  • Integrates with imaging for spatial context

• CRISPR-Based Epigenome Editing:

  • Targets histone modifying enzymes to regions with HIST1H2AG enrichment

  • Allows functional testing of variant-specific roles

  • Creates synthetic histone modification patterns

  • Measures consequences on chromatin accessibility and gene expression

• Mass Cytometry (CyTOF) Integration:

  • Labels HIST1H2AG antibody with rare metal isotopes

  • Enables simultaneous detection of dozens of histone marks

  • Provides single-cell resolution of histone variant patterns

  • Correlates with cellular phenotypes and states

• Cryo-Electron Microscopy Applications:

  • Uses HIST1H2AG antibody to identify specific nucleosomes

  • Determines structural consequences of histone variant incorporation

  • Visualizes interactions with chromatin remodeling complexes

  • Achieves near-atomic resolution of variant nucleosomes