HIST1H2AG (Ab-95) Antibody

What is HIST1H2AG and what cellular function does it serve?

HIST1H2AG (also known as H2AC11 or H2AFP) is a replication-dependent histone H2A type 1 protein that functions as a core component of nucleosomes. As part of the nucleosome structure, HIST1H2AG plays a central role in packaging and compacting DNA into chromatin, which limits DNA accessibility to cellular machineries requiring DNA as a template. This process is fundamental to transcription regulation, DNA repair, DNA replication, and maintaining chromosomal stability .

HIST1H2AG belongs to the family of canonical H2A proteins, which are encoded by multiple genes clustered in the genome. The protein's function is regulated through post-translational modifications that form part of the "histone code," which controls chromatin structure and accessibility .

What applications is the HIST1H2AG (Ab-95) antibody validated for?

The HIST1H2AG (Ab-95) antibody has been validated for several research applications, primarily ELISA (Enzyme-Linked Immunosorbent Assay) and IHC (Immunohistochemistry). For immunohistochemistry applications, the recommended dilution range is 1:20-1:200 .

This polyclonal antibody was raised in rabbit against a peptide sequence surrounding the Lysine 95 residue of human Histone H2A type 1. The antibody has been affinity-purified to ensure specificity and is supplied in a liquid form with preservatives (0.03% Proclin 300) and buffer (50% Glycerol, 0.01M PBS, pH 7.4) .

How does HIST1H2AG expression compare to other histone H2A variants?

Comparative expression analysis has revealed that HIST1H2AG exhibits significantly lower expression levels compared to other histone H2A variants. Studies in mouse models have shown that among the replication-dependent histone H2A genes, the expression level of HIST1H2AG was 10 to 30 times lower than that of HIST1H2AE, despite encoding identical proteins .

In a more comprehensive comparison among 13 genes in the Hist1 cluster, HIST1H2AE showed approximately 100 times higher expression than HIST1H2AA. These differential expression patterns suggest distinct regulatory mechanisms controlling histone variant expression, even within the same gene cluster .

What is the optimal protocol for histone extraction when studying HIST1H2AG?

For effective histone extraction when studying HIST1H2AG, the following acid extraction protocol has shown reliable results:

• Collect and pellet cells by centrifugation at 12,045 g for 15 minutes at 4°C

• Wash the pellet with 1 ml of TBS buffer (10 mM Tris–HCl (pH 7.4), 150 mM NaCl)

• Add 0.2 M sulfuric acid (H₂SO₄) to the washed pellet to extract histones

• Vortex thoroughly and incubate on ice for 30 minutes

• Centrifuge the solution at 12,045 g for 15 minutes at 4°C to remove cellular debris

• Collect the supernatant and add 80% acetone

• Precipitate overnight at -20°C

• Centrifuge the precipitated histones at 12,045 g for 15 minutes at 4°C

• Air-dry the pellet for 10 minutes and resuspend in HPLC-grade water

This protocol effectively isolates histones while preserving their native structure and post-translational modifications for subsequent analysis with the HIST1H2AG (Ab-95) antibody.

How should researchers optimize immunohistochemistry protocols when using HIST1H2AG (Ab-95) antibody?

When optimizing immunohistochemistry (IHC) protocols using the HIST1H2AG (Ab-95) antibody, researchers should consider the following methodological approach:

• Antibody dilution optimization: Begin with a dilution range of 1:20-1:200 as recommended . Perform a dilution series to determine optimal signal-to-noise ratio for your specific tissue type.

• Antigen retrieval: Since histones are tightly bound to DNA in chromatin, effective antigen retrieval is crucial. Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is recommended as a starting point.

• Blocking step: Use 5-10% normal serum from the same species as the secondary antibody for 1 hour at room temperature to reduce non-specific binding.

• Primary antibody incubation: Incubate with HIST1H2AG (Ab-95) antibody overnight at 4°C to ensure adequate binding to the target.

• Negative controls: Include sections without primary antibody to assess background staining.

• Positive controls: Include tissues known to express high levels of histone H2A proteins, such as proliferating tissues or cell lines with verified HIST1H2AG expression.

• Signal detection: Use a detection system appropriate for rabbit polyclonal antibodies, adjusting development time to achieve optimal signal intensity.

• Counterstaining: Use a light hematoxylin counterstain to visualize tissue architecture without obscuring the specific antibody signal.

For validation of staining patterns, compare results with known expression patterns of histone H2A variants in your tissue of interest.

How can ChIP-qPCR be effectively used with HIST1H2AG (Ab-95) antibody to study histone modifications?

Chromatin Immunoprecipitation followed by quantitative PCR (ChIP-qPCR) is a powerful technique for studying histone modifications and chromatin states associated with HIST1H2AG. Based on established methodologies used for histone H2A variants, the following protocol can be adapted for HIST1H2AG (Ab-95) antibody:

• Crosslinking: 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. Verify fragmentation by agarose gel electrophoresis.

• Immunoprecipitation: Incubate fragmented chromatin with HIST1H2AG (Ab-95) antibody (5-10 μg) overnight at 4°C. Include appropriate controls: IgG negative control and a positive control antibody (e.g., anti-histone H3).

• Washing and elution: Wash immunoprecipitated complexes to remove non-specific binding, then elute protein-DNA complexes.

• Reverse crosslinking: Reverse formaldehyde crosslinks by heating at 65°C for 4-6 hours.

• DNA purification: Purify the DNA using standard phenol-chloroform extraction or commercial kits.

• qPCR analysis: Design primers for regions of interest, including promoters of genes regulated by HIST1H2AG or sites known to be enriched for histone H2A variants.

When interpreting ChIP-qPCR results, it's important to note that histone H3 K9 acetylation levels in promoter regions correlate with gene expression levels of histone H2A variants. Studies have shown that highly expressed histone genes like H2AFZ and HIST3H2A have significantly higher H3 K9 acetylation compared to low-expression genes like HIST1H2AA .

What approaches can be used to study the functional differences between HIST1H2AG and other histone H2A variants?

To investigate functional differences between HIST1H2AG and other histone H2A variants, researchers can employ multiple complementary approaches:

• siRNA-mediated knockdown: Design specific siRNAs targeting HIST1H2AG and other H2A variants. This approach has been successfully used to demonstrate that different H2A isoforms have distinct effects on cell growth and proliferation. The specificity of knockdown can be verified using real-time PCR assays .

• Luciferase reporter assays: Clone the 5' UTR of HIST1H2AG and other histone H2A isoforms upstream of a luciferase reporter gene to compare their transcriptional regulation. This technique allows quantification of promoter activity and identification of regulatory elements specific to each variant .

• Chromatin dynamics analysis: Study incorporation rates of different histone variants into chromatin using tagged histones. Research on H2A variants has shown differential assembly into chromatin depending on transcriptional activity, with some variants (like H2A.B) being enriched in transcriptionally active regions .

• Cell cycle analysis: Monitor expression patterns throughout the cell cycle using synchronized cell populations. Replication-dependent histones like HIST1H2AG show characteristic expression patterns peaking in the middle of S-phase (2-4 hours after the beginning of S-phase) and decreasing toward the end (6 hours) .

• Epigenetic modification profiling: Compare the post-translational modification landscape associated with different H2A variants using mass spectrometry or modification-specific antibodies.

When examining the results, look for phenotypic changes in cell proliferation, transcription patterns, chromatin structure, and response to cellular stresses to determine functional specificity of HIST1H2AG compared to other H2A variants.

What are common sources of false positives or false negatives when using HIST1H2AG (Ab-95) antibody, and how can they be addressed?

When working with HIST1H2AG (Ab-95) antibody, researchers may encounter several sources of false results:

Common sources of false positives:

• Cross-reactivity with other H2A variants: Given the high sequence similarity among histone H2A family members, cross-reactivity can occur. HIST1H2AG shares identical amino acid sequences with several other H2A variants including HIST1H2AB, HIST1H2AC, HIST1H2AD, HIST1H2AE, HIST1H2AI, HIST1H2AN, and HIST1H2AO .

  Solution: Validate antibody specificity using knockout/knockdown controls. Perform western blot analysis with recombinant proteins of different H2A variants to assess cross-reactivity.

• Non-specific binding to highly basic proteins: Histones are highly basic proteins that can cause non-specific interactions.

  Solution: Use more stringent washing conditions and ensure adequate blocking. Consider including competing peptides not containing the target epitope.

Common sources of false negatives:

• Epitope masking by chromatin structure: The Lys-95 epitope might be masked by chromatin compaction or protein-protein interactions.

  Solution: Optimize chromatin preparation methods, including more effective nuclease digestion or sonication protocols. Try different antigen retrieval methods for IHC applications.

• Low abundance of HIST1H2AG: As shown in comparative studies, HIST1H2AG has relatively low expression compared to other H2A variants .

  Solution: Increase sample concentration, optimize antibody incubation conditions, or use more sensitive detection methods. Consider cell synchronization to capture peak expression during S-phase.

• Post-translational modifications affecting epitope recognition: Since the antibody targets a peptide sequence around Lys-95, modifications at or near this residue could interfere with antibody binding.

  Solution: When possible, assess the modification status of the histone preparation. Consider using antibodies targeting different epitopes of HIST1H2AG for confirmation.

How can researchers accurately quantify HIST1H2AG expression levels across different cell types or experimental conditions?

For accurate quantification of HIST1H2AG expression across different experimental conditions, researchers should implement the following comprehensive approach:

• RT-qPCR optimization:

  • Design primers specific to HIST1H2AG, carefully distinguishing it from other highly similar H2A variants

  • Validate primer specificity through melt curve analysis and sequencing of PCR products

  • Use multiple reference genes for normalization, selecting those with stable expression across your experimental conditions

  • Include standard curves to determine absolute quantification where needed

• Western blot quantification:

  • Extract histones using the acid extraction protocol described earlier

  • Load equal amounts of total histone protein (10-20 μg recommended)

  • Include recombinant HIST1H2AG standards at known concentrations for quantification

  • Use fluorescence-based detection systems rather than chemiluminescence for more accurate quantification

  • Perform densitometry using software that can correct for background and saturation

• Cell synchronization for temporal studies:

  • Since histone expression fluctuates throughout the cell cycle, synchronize cells using established methods

  • Collect samples at defined time points throughout S-phase (0, 1, 2, 3, 4, 5, and 6 hours) to capture expression dynamics

  • Verify synchronization efficiency using flow cytometry

• Controls and normalizations:

  • Include both constitutively expressed histones (like H2AFZ) and other replication-dependent histones for comparison

  • For ChIP-qPCR studies, normalize to input DNA and use both positive genomic regions (active genes) and negative controls (silenced regions like γ-satellite)

• Data analysis considerations:

  • Apply appropriate statistical tests based on your experimental design

  • Consider using the ΔΔCt method for RT-qPCR with appropriate validation

  • For absolute quantification, generate standard curves using purified HIST1H2AG mRNA or recombinant protein

How is HIST1H2AG being studied in relation to cancer epigenetics and potential therapeutic targets?

The study of HIST1H2AG in cancer epigenetics represents an emerging area of research, particularly in light of the discovery of "oncohistones" - mutated histones that can drive tumorigenesis. While specific information on HIST1H2AG in cancer is limited in the provided search results, the broader context of histone H2A variants in cancer provides valuable insights:

• Potential role in cancer epigenetics:

  • Histone mutations, particularly those affecting post-translational modification sites, have been identified in pediatric brain tumors, chondroblastoma, and giant cell tumor of bone .

  • These mutations often affect residues normally targeted by post-translational modifications in the histone N-terminal tails, potentially interfering with epigenetic regulation .

  • Research into HIST1H2AG should focus on identifying any cancer-specific mutations and their functional consequences on chromatin structure and gene expression.

• Differential expression in cancer tissues:

  • Studies comparing histone variant expression between normal and tumor tissues may reveal dysregulation of HIST1H2AG in specific cancer types.

  • Analyzing The Cancer Genome Atlas (TCGA) and other cancer databases for alterations in HIST1H2AG expression could identify cancer types where this histone variant plays a significant role.

• Epigenetic targeting strategies:

  • As histone modifications regulate the expression of histone genes themselves (e.g., the correlation between H3 K9 acetylation and histone gene expression) , targeting these modifications could potentially modulate HIST1H2AG expression.

  • Investigating the interaction between HIST1H2AG and chromatin-modifying enzymes may reveal novel therapeutic targets for cancers with aberrant chromatin states.

• Functional consequences of altered HIST1H2AG expression:

  • Studies using siRNA knockdown approaches have demonstrated that alterations in specific histone H2A isoforms can affect cell growth and proliferation .

  • Similar approaches could determine whether modulating HIST1H2AG levels affects cancer cell phenotypes such as proliferation, invasion, or drug resistance.

Future research should explore HIST1H2AG-specific mutations in cancer, their effects on chromatin structure and gene expression patterns, and potential therapeutic interventions targeting these alterations.

What role does HIST1H2AG play in regulating chromatin dynamics during cellular stress or DNA damage response?

Understanding the role of HIST1H2AG in chromatin dynamics during cellular stress or DNA damage requires integration of histone H2A variant biology with stress response mechanisms:

• Chromatin remodeling during stress responses:

  • Histone H2A variants play crucial roles in regulating chromatin structure and accessibility during stress responses and DNA damage repair.

  • While specific data on HIST1H2AG is limited, studies on related H2A variants provide a framework for investigation. For example, H2A.B becomes enriched in transcriptionally active and dynamic viral chromatin, suggesting that specific H2A variants can be preferentially incorporated to promote chromatin dynamics .

• Methodological approaches for studying HIST1H2AG in stress responses:

  • Live-cell imaging: Using fluorescently tagged HIST1H2AG to track its dynamics during cellular stress in real-time.

  • ChIP-seq analysis: Before and after stress induction to map genome-wide redistribution of HIST1H2AG.

  • Mass spectrometry: To identify stress-induced post-translational modifications specific to HIST1H2AG.

  • Proximity labeling: To identify stress-specific interaction partners of HIST1H2AG.

• Integration with DNA damage response pathways:

  • Investigating potential interactions between HIST1H2AG and key DNA damage response proteins.

  • Examining how depletion of HIST1H2AG affects cellular sensitivity to genotoxic agents or radiation.

  • Analyzing the dynamics of HIST1H2AG at DNA damage sites using laser microirradiation coupled with live-cell imaging.

• Cell-cycle dependent responses:

  • As a replication-dependent histone, HIST1H2AG expression peaks during S-phase . This timing coincides with periods of potential replication stress.

  • Studying how replication stress specifically affects HIST1H2AG deposition and function could reveal specialized roles in preserving genomic integrity during DNA replication.

Research into these aspects would provide valuable insights into the specific contributions of HIST1H2AG to stress responses and DNA damage repair, potentially revealing novel therapeutic opportunities in contexts where these processes are dysregulated.

How does the expression of HIST1H2AG compare to other H2A variants across different tissue types and developmental stages?

Comparative analysis of histone H2A variant expression, including HIST1H2AG, reveals significant differences across tissues and developmental contexts:

The expression pattern of HIST1H2AG, like other replication-dependent histones, shows a characteristic increase from the beginning of S-phase (0h) to the middle (2-4h), followed by a decrease toward the end (6h) . This contrasts with replication-independent variants like H2AFZ, which lack such pronounced S-phase peaks.

Interestingly, even histones that encode identical proteins show dramatically different expression levels. HIST1H2AG encodes the same protein as seven other H2A genes (HIST1H2AB, 2AC, 2AD, 2AE, 2AI, 2AN, and 2AO), yet its expression is 10-30 times lower than HIST1H2AE . This suggests complex regulatory mechanisms controlling histone variant expression beyond simple protein function.

The differences in expression levels correlate with histone H3 K9 acetylation levels in the promoter regions. Highly expressed genes like H2AFZ and HIST3H2A show significantly higher H3 K9 acetylation compared to low-expression genes like HIST1H2AA . This epigenetic regulatory mechanism likely applies to HIST1H2AG as well, potentially explaining its relatively low expression level.

What are the key technical differences between using HIST1H2AG (Ab-95) antibody and other commercial antibodies targeting related histone H2A variants?

When comparing HIST1H2AG (Ab-95) antibody with other antibodies targeting histone H2A variants, researchers should consider several technical aspects that impact experimental design and result interpretation:

When designing experiments using HIST1H2AG (Ab-95) antibody, researchers should:

• Validate specificity: Due to the high sequence similarity among H2A variants, rigorous validation using knockdown/knockout controls is essential.

• Optimize protocols for each application: While validated for ELISA and IHC, adaptation to other techniques requires careful optimization.

• Consider epitope accessibility: The Lys-95 epitope may have different accessibility depending on chromatin state and experimental conditions.

• Account for expression level differences: Since HIST1H2AG is expressed at relatively low levels compared to other H2A variants , detection methods may need to be more sensitive.

• Include appropriate controls: Use both negative controls (IgG, irrelevant antibodies) and positive controls (pan-H2A antibodies) to contextualize HIST1H2AG-specific signals.

By carefully considering these technical differences, researchers can select the most appropriate antibody for their specific experimental questions and properly interpret the resulting data.