HIST1H2AG (Ab-101) Antibody

What is HIST1H2AG and what are its cellular functions?

HIST1H2AG is a member of the histone H2A family, specifically a replication-dependent (RD) variant. It belongs to the histone cluster 1 and is also known by several aliases including H2A.1, H2AFP, H2AFC, H2AFD, H2AFI, and H2AFN . This protein functions as a core component of nucleosomes, the basic unit of chromatin packaging in eukaryotic cells. HIST1H2AG participates in DNA packaging, chromatin remodeling, and plays critical roles in epigenetic regulation of gene expression.

The protein is involved in multiple cellular processes including:

• Chromatin organization and higher-order structure formation

• Regulation of DNA transcription

• DNA damage response and repair mechanisms

• Cell cycle progression

• Maintenance of genome stability

HIST1H2AG participates in epigenetic and nuclear signaling pathways, making it a significant target for research involving gene expression regulation, development, and disease processes .

What are the validated applications for HIST1H2AG (Ab-101) Antibody?

The HIST1H2AG (Ab-101) Antibody has been validated for multiple research applications:

The antibody has been successfully used for detecting HIST1H2AG in paraffin-embedded human tissue samples including breast cancer tissue and glioma tissue as demonstrated by immunohistochemical analyses . Immunofluorescent analysis has also been performed in HeLa cells, confirming the antibody's effectiveness in cellular localization studies .

What are the key specifications of the HIST1H2AG (Ab-101) Antibody?

This polyclonal antibody targets a specific epitope around the Thr-101 site of human Histone H2A type 1, making it highly specific for research applications focused on this particular protein and its modifications .

How should I optimize immunohistochemistry protocols with this antibody?

For optimal immunohistochemistry results with HIST1H2AG (Ab-101) Antibody, follow these methodological guidelines:

Sample Preparation:

• Fix tissues in 10% neutral buffered formalin for 24-48 hours at room temperature

• Process and embed in paraffin using standard procedures

• Section tissues at 4-6 μm thickness

• Mount sections on positively charged slides

Antigen Retrieval Protocol:

• Deparaffinize sections in xylene and rehydrate through graded ethanol series

• Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0)

• Heat sections at 95-100°C for 15-20 minutes in a water bath or pressure cooker

• Allow slides to cool to room temperature in the retrieval solution (approximately 20 minutes)

Staining Protocol:

• Block endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes

• Block non-specific binding with 5% normal serum in PBS for 1 hour

• Incubate with HIST1H2AG (Ab-101) Antibody at dilutions between 1:20-1:200 overnight at 4°C

• Wash 3 times with PBS (5 minutes each)

• Apply appropriate secondary antibody and develop according to standard detection protocols

• Counterstain, dehydrate, clear, and mount

For nuclear antigens like HIST1H2AG, ensure proper nuclear permeabilization and optimize antibody concentration through titration experiments. The antibody has been successfully used to detect HIST1H2AG in human breast cancer and glioma tissues, showing clear nuclear localization patterns .

What controls should be included when working with this antibody?

Incorporating appropriate controls is essential for validating experimental results with HIST1H2AG (Ab-101) Antibody:

Positive Controls:

• Human breast cancer tissue sections (demonstrated positive reactivity)

• Human glioma tissue sections (demonstrated positive reactivity)

• HeLa cells for immunofluorescence (demonstrated positive reactivity)

• Cell lines known to express HIST1H2AG based on RNA or protein expression databases

Negative Controls:

• Primary Antibody Omission Control: Replace primary antibody with antibody diluent to assess non-specific binding of detection reagents

• Isotype Control: Use normal rabbit IgG at the same concentration as the primary antibody

• Absorption Control: Pre-incubate the antibody with excess immunizing peptide prior to staining

• Tissue-Negative Control: Include tissues known to express low/no levels of HIST1H2AG

Technical Controls:

• Dilution Series: Test a range of antibody dilutions (1:20, 1:50, 1:100, 1:200) to determine optimal signal-to-noise ratio

• Antigen Retrieval Comparison: Compare different antigen retrieval methods to optimize epitope accessibility

• Detection System Control: Include a well-characterized antibody targeting a different nuclear protein using the same detection method

Documenting and reporting these controls enhances experimental rigor and reproducibility, helping to validate the specificity of observed HIST1H2AG signals .

How can I optimize immunofluorescence protocols with this antibody?

For high-quality immunofluorescence results using HIST1H2AG (Ab-101) Antibody, consider the following optimization strategy:

Cell Preparation:

• Culture cells on coverslips or chamber slides to 70-80% confluence

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

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

• Block with 5% normal serum and 0.3% Triton X-100 in PBS for 1 hour

Staining Protocol:

• Dilute HIST1H2AG (Ab-101) Antibody to 1:50-1:200 in blocking buffer

• Incubate overnight at 4°C in a humidified chamber

• Wash 3 times with PBS (5 minutes each)

• Apply fluorophore-conjugated secondary antibody (anti-rabbit) at 1:500-1:1000 for 1 hour at room temperature

• Wash 3 times with PBS (5 minutes each)

• Counterstain nuclei with DAPI (1 μg/mL) for 5 minutes

• Mount with anti-fade mounting medium

Optimization Parameters:

• Test multiple fixation methods (paraformaldehyde, methanol, or acetone)

• Adjust permeabilization time (5-15 minutes) and detergent concentration (0.1-0.3%)

• Vary antibody concentration in the recommended range (1:50-1:200)

• Modify incubation times and temperatures

• Compare different antifade mounting media to preserve fluorescence

The antibody has been successfully used for immunofluorescent analysis in HeLa cells, showing distinct nuclear staining patterns consistent with histone localization .

How can HIST1H2AG (Ab-101) Antibody be used in studies of epigenetic modifications?

HIST1H2AG (Ab-101) Antibody is valuable for investigating epigenetic mechanisms due to the central role of histones in chromatin regulation:

Chromatin Immunoprecipitation (ChIP) Applications:

• Optimize crosslinking conditions (1% formaldehyde for 10 minutes is standard)

• Sonicate chromatin to 200-500 bp fragments

• Use 2-5 μg of HIST1H2AG (Ab-101) Antibody per immunoprecipitation

• Include appropriate input and IgG controls

• Analyze by qPCR, sequencing, or array-based methods

Co-Immunoprecipitation for Histone Modification Studies:

• Use nuclear extracts from cells of interest

• Immunoprecipitate with HIST1H2AG (Ab-101) Antibody

• Analyze co-precipitated proteins or histone modifications by western blotting

• Probe for known histone modifiers or readers that interact with H2A variants

Epigenetic Profiling:

• Combine with antibodies against histone marks (H3K9me3, H3K27me3, H4K16ac) to investigate correlation between HIST1H2AG occupancy and specific modifications

• Study HIST1H2AG distribution in relation to heterochromatin/euchromatin boundaries

• Investigate changes in HIST1H2AG localization during aging or disease processes

Research has revealed important connections between H2A variants and aging processes. For example, changes in histone modifications like H3K56Ac and H4K16Ac have been linked to lifespan regulation, with implications for how H2A variants might interact with these modified residues in aging cells .

What are common causes of background staining and how can they be addressed?

Background staining can complicate interpretation of HIST1H2AG (Ab-101) Antibody results. Here are common causes and troubleshooting approaches:

When troubleshooting, modify one parameter at a time and document results systematically. For this antibody, nuclear localization is expected, so cytoplasmic staining may indicate non-specific binding that needs to be addressed through protocol optimization .

How does HIST1H2AG relate to H2A.Z isoforms in research contexts?

HIST1H2AG is a canonical histone H2A variant, which differs from the specialized histone H2A.Z variants that play critical regulatory roles:

Relationship to H2A.Z Isoforms:
HIST1H2AG belongs to the replication-dependent (RD) H2A family, while H2A.Z is a replication-independent (RI) variant. H2A.Z further exists as two major isoforms (H2A.Z-1 and H2A.Z-2) that differ by only a few amino acids but appear to have distinct biological functions .

Functional Comparisons:

• Differential Expression: While canonical H2A variants like HIST1H2AG are expressed primarily during S-phase, H2A.Z variants are expressed throughout the cell cycle

• Chromatin Localization: H2A.Z variants are often enriched at promoters and enhancers, while canonical H2A is more uniformly distributed

• Transcriptional Roles: H2A.Z has been implicated in both gene activation and repression in a context-dependent manner

Research Applications:

• Comparative ChIP-seq experiments can reveal differential genomic distributions between HIST1H2AG and H2A.Z variants

• Co-immunoprecipitation studies can identify differential protein interactions

• Expression analyses during development or disease progression can reveal distinct patterns

Disease Relevance:
The two H2A.Z isoforms have demonstrated different roles in cancer contexts:

• H2A.Z-1 shows increased expression in prostate cancer in response to androgen treatment

• H2A.Z-1 plays a regulatory role in epithelial-mesenchymal transition in liver cancer

• H2A.Z-2 has been identified as a driver of malignant melanoma

• H2A.Z-2 is involved in DNA damage response, particularly relevant in UV-induced DNA damage cases

Understanding the distinct biological roles of these related histone variants provides important context for researchers investigating HIST1H2AG function.

How should immunohistochemistry results with HIST1H2AG (Ab-101) be quantified?

Quantification of HIST1H2AG immunohistochemistry requires standardized approaches for reliable data interpretation:

Recommended Quantification Methods:

• H-Score Method:

  • Assess both staining intensity and percentage of positive cells

  • Score intensity as 0 (negative), 1+ (weak), 2+ (moderate), or 3+ (strong)

  • Calculate H-score = (1 × % of 1+ cells) + (2 × % of 2+ cells) + (3 × % of 3+ cells)

  • Final score ranges from 0-300

• Allred Scoring System:

  • Combine proportion score (0-5) and intensity score (0-3)

  • Proportion: 0=none, 1=<1%, 2=1-10%, 3=11-33%, 4=34-66%, 5=67-100%

  • Intensity: 0=negative, 1=weak, 2=moderate, 3=strong

  • Total score: sum of proportion and intensity (0-8)

• Digital Image Analysis:

  • Capture standardized digital images of stained sections

  • Use software like ImageJ, QuPath or commercial platforms

  • Measure parameters including:

    • Nuclear positivity percentage

    • Optical density (mean, integrated)

    • Staining intensity distribution

Standardization Recommendations:

• Analyze at least 5 random high-power fields per sample

• Include at least 1000 cells in total counting

• Use batch analysis when processing multiple samples

• Include positive and negative controls in every quantification session

• Blind the scorer to experimental conditions when possible

• For clinical samples, have at least two independent scorers

When reporting results, clearly describe the quantification method, number of fields/cells analyzed, and statistical approaches used. Include representative images showing the range of staining patterns observed .

How can HIST1H2AG expression be correlated with other histone modifications?

Correlating HIST1H2AG expression with other histone modifications provides valuable insights into epigenetic regulation mechanisms:

Multiparameter Analysis Approaches:

• Sequential Immunostaining:

  • Perform multiple rounds of staining on the same section

  • Use digital image registration to correlate signals

  • Apply spectral unmixing for multiplexed fluorescence

  • Examples of relevant modifications to correlate: H3K9me3, H3K27me3, H4K16ac, H3K56ac

• Dual Immunofluorescence:

  • Simultaneously detect HIST1H2AG and other histone marks

  • Use species-specific or isotype-specific secondary antibodies

  • Analyze co-localization using Pearson's or Manders' coefficients

  • Visualize spatial relationships using intensity correlation plots

• Integrative Data Analysis:

  • Combine IHC/IF data with ChIP-seq or cut&run results

  • Correlate protein expression with genomic occupancy patterns

  • Map relationships between HIST1H2AG and modifications at specific genomic loci

Relevant Histone Modifications for Correlation Studies:
Research indicates several histone modifications have functional relationships with H2A variants and aging processes:

Research has shown that histone modifications undergo progressive changes during aging, with implications for chromatin structure and gene expression regulation. Correlating HIST1H2AG with these modifications can provide insights into its role in age-related chromatin changes .

What is the significance of HIST1H2AG in cancer research?

HIST1H2AG and related histone variants have emerging roles in cancer biology that make them valuable research targets:

Cancer-Related Functions:
While specific HIST1H2AG functions in cancer are still being elucidated, research on related H2A variants provides important context:

• Altered Expression in Cancer:

  • H2A.Z isoforms show differential expression across cancer types

  • H2A.Z-1 demonstrates increased expression in prostate cancer following androgen treatment

  • The H2A.Z-1 promoter contains MYC binding elements, suggesting regulation by this oncogenic transcription factor

  • H2A.Z-1 has been implicated in epithelial-mesenchymal transition in liver cancer

• Role in Malignant Transformation:

  • H2A.Z-2 has been identified as a driver of malignant melanoma

  • This isoform influences cellular proliferation and drug sensitivity in melanoma

  • The effect may be related to H2A.Z-2's function in DNA damage response, particularly relevant for UV-induced damage

  • Post-translational modifications like sumoylation regulate H2A.Z-2 exchange at DNA damage sites

Research Applications in Cancer Studies:

• Use HIST1H2AG (Ab-101) Antibody to evaluate expression patterns across cancer types

• Correlate expression with clinical parameters and patient outcomes

• Investigate changes in genomic distribution during malignant transformation

• Study potential regulatory relationships with known oncogenes and tumor suppressors

The antibody has been validated for use in breast cancer and glioma tissue samples, making it a valuable tool for investigating HIST1H2AG's role in these cancer types .

How is HIST1H2AG involved in epigenetic regulation during development and aging?

HIST1H2AG and related histone variants play critical roles in epigenetic regulation throughout the lifespan:

Developmental Regulation:
Research on H2A variants suggests important developmental functions:

• H2A.Z isoforms have differential, context-specific roles in neuronal activity-induced transcription

• H2A.Z-2 has been implicated in craniofacial development, with defects in this variant associated with floating-harbor developmental syndrome (FHS)

• Hippocampal levels of H2A.Z-2 are affected by fetal alcohol spectrum disorder (FASD)

• The functional involvement of histone variant isoforms appears particularly important during development

Epigenetic Changes During Aging:
Histone variants and their modifications undergo significant changes during aging:

• Chromatin Architecture Changes:

  • Progressive loss of heterochromatin and more relaxed chromatin structure with age

  • Alterations in histone modification patterns, including:

    • Decrease in H3K56ac and H3K36me3

    • Increase in H4K16ac and H3K27me3

    • Changes in H3K9me3 that vary by tissue type

• Regulatory Mechanisms:

  • Sirtuins (particularly Sir2) regulate H4K16ac levels, with implications for aging

  • Overexpression of Sir2 extends lifespan while increased H4K16ac promotes aging

  • The chromatin structure at telomeres is particularly affected by these changes

• Tissue-Specific Patterns:

  • Different tissues show distinct patterns of histone modification changes during aging

  • Brain tissues may exhibit different patterns compared to dividing cells

  • Analysis of senescence-accelerated mouse brain tissues revealed decreased H4K20me1 and increased H3K27me3

Understanding HIST1H2AG's role in these processes could provide valuable insights into fundamental mechanisms of development and aging, with potential implications for age-related diseases.

What methodologies are recommended for studying HIST1H2AG in chromatin regulation?

Advanced methodological approaches for investigating HIST1H2AG's role in chromatin regulation include:

Chromatin Structure Analysis:

• ATAC-seq (Assay for Transposase-Accessible Chromatin):

  • Compare chromatin accessibility in systems with normal vs. altered HIST1H2AG levels

  • Correlate accessibility with HIST1H2AG occupancy determined by ChIP-seq

  • Analyze changes in accessibility following experimental manipulation of HIST1H2AG

• Hi-C and Micro-C:

  • Map three-dimensional chromatin interactions

  • Identify topologically associating domains (TADs) containing HIST1H2AG

  • Assess how HIST1H2AG influences higher-order chromatin organization

• CUT&RUN or CUT&Tag:

  • More sensitive alternatives to traditional ChIP for mapping HIST1H2AG genomic distribution

  • Particularly useful for samples with limited material

  • Can be combined with sequential approaches to map multiple factors

Functional Analysis:

• CRISPR-Cas9 Approaches:

  • Generate HIST1H2AG knockout or knockdown models

  • Create specific mutations at post-translational modification sites

  • Employ CRISPRi/CRISPRa for targeted modulation of expression

• Mass Spectrometry Analysis:

  • Identify HIST1H2AG interaction partners through AP-MS

  • Map post-translational modifications by MS/MS

  • Quantify changes in modification patterns across conditions

• Live-Cell Imaging:

  • Use fluorescently tagged HIST1H2AG to track dynamics

  • Apply FRAP (Fluorescence Recovery After Photobleaching) to assess exchange rates

  • Implement super-resolution microscopy for detailed localization

Integrative Bioinformatic Analysis:

• Correlate HIST1H2AG occupancy with transcriptional activity and other epigenetic marks

• Perform motif analysis to identify sequence preferences for HIST1H2AG deposition

• Utilize machine learning approaches to predict HIST1H2AG distribution and functional impacts

When designing these experiments, researchers should carefully consider the specificity of HIST1H2AG (Ab-101) Antibody and incorporate appropriate controls to validate findings. The antibody's specificity for the Thr-101 region makes it particularly valuable for studies focusing on this domain and its potential modifications .