HIST1H2AG (Ab-74) Antibody

What is HIST1H2AG and what is its role in cellular processes?

HIST1H2AG is a histone H2A variant, specifically Histone H2A type 1, that functions as a core component of nucleosomes. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to cellular machineries requiring DNA as a template. HIST1H2AG and other histones play central roles in transcription regulation, DNA repair, DNA replication, and chromosomal stability . DNA accessibility is regulated through a complex set of post-translational modifications of histones (the "histone code") and nucleosome remodeling. HIST1H2AG is primarily localized in the nucleus and chromosome .

What are the optimal storage conditions for HIST1H2AG (Ab-74) antibody?

For short-term storage (up to 1 week), store HIST1H2AG (Ab-74) antibody at +4°C. For long-term storage, aliquot and store at -20°C or -80°C . It's critical to avoid repeated freeze-thaw cycles as each cycle can result in the loss of approximately half of the antibody's binding activity . The antibody is typically supplied in phosphate-buffered saline (pH 7.4) containing 0.03% Proclin and 50% Glycerol . When planning experiments, consider the stability profile of the antibody to ensure optimal performance.

What experimental applications is HIST1H2AG (Ab-74) antibody validated for?

The HIST1H2AG (Ab-74) antibody has been validated for multiple experimental applications including Enzyme-Linked Immunosorbent Assay (ELISA), Western Blotting (WB), and Immunoprecipitation (IP) . For Western Blotting, the recommended dilution range is 1:100-1:1000, while for Immunoprecipitation, the recommended dilution range is 1:200-1:2000 . The predicted molecular weight of the target protein is 15 KDa, which has been confirmed by experimental observation . When designing experiments, consider the optimal dilution based on your specific experimental conditions and detection methods.

How can I optimize experimental conditions for Western blotting with HIST1H2AG (Ab-74) antibody?

For optimal Western blotting with HIST1H2AG (Ab-74) antibody, begin with these methodological considerations:

• Sample preparation: Use freshly prepared nuclear extracts as HIST1H2AG is primarily localized in the nucleus. Consider using phosphatase and protease inhibitors to preserve post-translational modifications.

• Dilution optimization: Start with the middle of the recommended range (1:500) for Western blotting . Adjust based on signal intensity and background levels.

• Blocking optimization: Use 5% non-fat dry milk or BSA in TBS-T. Given that the antibody is derived from rabbit host and reacts with human and rat species, ensure appropriate secondary antibody selection .

• Control selection: Include positive controls (human or rat cell lines with known HIST1H2AG expression) and negative controls (tissues or cell lines where the target is not expressed).

• Detection system: For the 15 KDa target, use a detection system with appropriate sensitivity for low molecular weight proteins .

• Stripping and reprobing: If examining post-translational modifications, consider that harsh stripping conditions may affect epitope recognition in subsequent probing.

What methodological approaches can overcome cross-reactivity issues with HIST1H2AG (Ab-74) antibody?

Cross-reactivity can be a significant challenge when working with histone antibodies due to the high sequence homology between histone variants. To overcome potential cross-reactivity with HIST1H2AG (Ab-74) antibody:

• Pre-absorption testing: Perform pre-absorption tests with recombinant HIST1H2AG and related histone proteins to assess specificity.

• Peptide competition assay: Conduct peptide competition assays using the immunogen peptide (sequence around site of Lys-74 derived from Human Histone H2A type 1) .

• Knockout/knockdown validation: Validate specificity using HIST1H2AG knockout or knockdown models to confirm antibody specificity.

• Orthogonal verification: Confirm results using alternative methods such as mass spectrometry or ChIP-seq with different antibodies targeting the same protein.

• Stringent washing conditions: Optimize washing steps in immunoassays to reduce non-specific binding.

• Species-specific considerations: Remember that this antibody is reactive to human and rat species, so choose appropriate experimental models .

What are the best approaches for detecting HIST1H2AG post-translational modifications?

Detecting post-translational modifications (PTMs) of HIST1H2AG requires specialized methodological approaches:

• Modification-specific antibodies: Use antibodies specifically targeting known PTMs of HIST1H2AG in combination with the Ab-74 antibody.

• Sample preparation: Preserve PTMs during extraction by using phosphatase inhibitors, deacetylase inhibitors, and protease inhibitors.

• Mass spectrometry: Employ LC-MS/MS following immunoprecipitation with HIST1H2AG (Ab-74) antibody to identify and quantify PTMs.

• Sequential ChIP: Perform sequential chromatin immunoprecipitation to determine co-occurrence of different modifications.

• Proximity ligation assay: Use proximity ligation assays to detect interactions between HIST1H2AG and modifying enzymes or reader proteins.

• 2D gel electrophoresis: Employ 2D gel electrophoresis followed by Western blotting to separate HIST1H2AG isoforms with different PTMs.

These approaches provide complementary information about the complex regulatory landscape of histone modifications that contribute to the histone code regulating DNA accessibility .

How can HIST1H2AG (Ab-74) antibody be used to investigate cancer mechanisms?

HIST1H2AG has been found altered in hepatocellular carcinoma and colon cancer , making it a valuable target for cancer research. Advanced methodological approaches include:

• ChIP-seq profiling: Use chromatin immunoprecipitation followed by sequencing to map genome-wide distribution of HIST1H2AG in normal versus cancer tissues.

• Tissue microarray analysis: Employ the antibody in immunohistochemistry on tissue microarrays containing multiple cancer types to correlate expression with clinical parameters.

• Cancer cell line panels: Screen HIST1H2AG expression across cancer cell line panels to identify cancer-specific alterations.

• Functional studies: Combine antibody-based detection with CRISPR-Cas9 knockout or overexpression studies to establish functional roles in cancer progression.

• Drug response correlation: Investigate changes in HIST1H2AG localization or PTMs after treatment with epigenetic drugs to identify potential biomarkers of response.

• Patient-derived xenograft models: Analyze HIST1H2AG expression and localization in PDX models to understand its role in tumor growth and metastasis.

These approaches can provide insights into how alterations in histone variants contribute to cancer development and progression .

What is the role of HIST1H2AG in immune regulation and how can it be studied?

While the search results don't directly address HIST1H2AG in immune regulation, research on histone H2A variants indicates potential immunological relevance. H2A-reactive B cells have been identified as functionally anergic in healthy individuals but can be activated under certain conditions . Methodological approaches include:

• B cell repertoire analysis: Isolate B cells reactive to HIST1H2AG and characterize their antibody repertoire.

• Tolerance studies: Investigate how HIST1H2AG-reactive B cells are regulated by peripheral tolerance mechanisms.

• TLR stimulation experiments: Examine how Toll-like receptor stimulation affects HIST1H2AG-reactive B cell activation and antibody production .

• T cell help assays: Study how T cell help influences HIST1H2AG-reactive B cell responses and tolerance breaking .

• Flow cytometric analysis: Use flow cytometry to characterize HIST1H2AG-reactive B cells and their expression of inhibitory mediators like CD5 and PTEN .

• Calcium mobilization assays: Assess functional anergy by measuring calcium mobilization upon immunoreceptor stimulation .

These approaches can elucidate the complex relationship between histone variants and immune regulation.

How can HIST1H2AG (Ab-74) antibody be used to study nucleosome dynamics and chromatin remodeling?

Studying nucleosome dynamics and chromatin remodeling with HIST1H2AG (Ab-74) antibody requires sophisticated methodological approaches:

• FRAP (Fluorescence Recovery After Photobleaching): Use fluorescently tagged HIST1H2AG to study dynamics in living cells, complemented by antibody validation in fixed cells.

• Single-molecule tracking: Combine with super-resolution microscopy to track individual HIST1H2AG molecules in the nucleus.

• ATAC-seq correlation: Correlate HIST1H2AG occupancy (via ChIP-seq) with chromatin accessibility (via ATAC-seq) to understand its role in chromatin compaction.

• Nucleosome occupancy mapping: Use MNase-seq in conjunction with HIST1H2AG ChIP to map variant-specific nucleosome positioning.

• Protein interaction studies: Perform immunoprecipitation with HIST1H2AG (Ab-74) antibody followed by mass spectrometry to identify interaction partners involved in chromatin remodeling.

• In vitro nucleosome assembly: Reconstitute nucleosomes with recombinant HIST1H2AG to study its effect on nucleosome stability and DNA accessibility.

These approaches provide insights into how HIST1H2AG contributes to the dynamic regulation of chromatin structure and function .

How should researchers interpret unexpected molecular weight variations when detecting HIST1H2AG?

When encountering unexpected molecular weight variations in HIST1H2AG detection:

• Expected versus observed weight: The predicted molecular weight of HIST1H2AG is 15 KDa, which should match the observed band size in Western blots . Significant deviations warrant investigation.

• Post-translational modifications: Higher molecular weight bands may indicate ubiquitination, SUMOylation, or other modifications that add substantial mass.

• Proteolytic cleavage: Lower molecular weight bands could result from proteolytic cleavage during sample preparation. Ensure proper use of protease inhibitors.

• Splice variants: Consider the possibility of detecting splice variants, though not specifically reported for HIST1H2AG in the search results.

• Denaturation conditions: Insufficient denaturation can cause anomalous migration. Ensure complete denaturation with appropriate SDS concentration and heating.

• Cross-reactivity: Evaluate potential cross-reactivity with other histone H2A variants with similar molecular weights, especially those listed as alternate names (HIST1H2AI, HIST1H2AK, HIST1H2AL, HIST1H2AM, H2AFP, H2AFC, H2AFD, H2AFI, H2AFN) .

Careful validation through complementary methods is essential for accurate interpretation of unexpected molecular weight variations.

What are the common pitfalls in ChIP experiments using HIST1H2AG (Ab-74) antibody and how can they be addressed?

Common pitfalls in ChIP experiments with HIST1H2AG (Ab-74) antibody include:

• Epitope masking: If the Lys-74 epitope is masked by protein-protein interactions or modifications, it can reduce antibody recognition. Use alternative fixation conditions or enzymatic treatments to improve accessibility.

• Crosslinking efficiency: Optimize formaldehyde concentration and crosslinking time for histone proteins, which typically require milder conditions than transcription factors.

• Sonication parameters: Adjust sonication conditions to achieve optimal chromatin fragmentation (200-500 bp) for histone ChIP.

• Antibody specificity: Validate specificity in the context of crosslinked chromatin using peptide competition or HIST1H2AG-depleted chromatin.

• Input normalization: Ensure proper input normalization, especially when comparing samples with different chromatin accessibility.

• IP efficiency: Optimize antibody concentration for immunoprecipitation (recommended range: 1:200-1:2000) and consider using protein A beads for rabbit IgG antibodies.

Addressing these pitfalls through careful optimization and validation is crucial for generating reliable ChIP data with HIST1H2AG (Ab-74) antibody.

How can researchers distinguish between distinct H2A variants in experimental settings?

Distinguishing between highly homologous H2A variants requires specific methodological approaches:

• Epitope selection: The HIST1H2AG (Ab-74) antibody targets the region around Lys-74 , which may differ among H2A variants. Compare epitope sequences across variants to predict potential cross-reactivity.

• Western blot optimization: Use high-resolution SDS-PAGE systems (e.g., Tricine-SDS-PAGE) to separate closely related histone variants with subtle mass differences.

• Isoelectric focusing: Employ 2D gel electrophoresis with isoelectric focusing to separate variants based on charge differences.

• Mass spectrometry: Use targeted mass spectrometry approaches like Multiple Reaction Monitoring (MRM) to distinguish between variants based on unique peptide signatures.

• Variant-specific knockdown: Perform siRNA-mediated knockdown of specific variants to confirm antibody specificity.

• Recombinant protein controls: Include recombinant proteins of different H2A variants as controls in immunoblotting experiments.

These approaches are particularly important given the number of related H2A variants (HIST1H2AG, HIST1H2AI, HIST1H2AK, HIST1H2AL, HIST1H2AM) with high sequence similarity .

How is HIST1H2AG expression altered in different cancer types and what methodologies best capture these changes?

Alterations in HIST1H2AG have been documented in hepatocellular carcinoma and colon cancer . To comprehensively analyze expression changes across cancer types:

• Multi-omics integration: Combine antibody-based detection with RNA-seq and proteomics data to correlate transcriptional and post-transcriptional regulation.

• Cancer-specific tissue microarrays: Develop tissue microarrays representing multiple stages of cancer progression to track expression changes during tumorigenesis.

• Patient-derived organoids: Use HIST1H2AG (Ab-74) antibody in immunofluorescence studies of patient-derived organoids to preserve tumor heterogeneity.

• Liquid biopsy analysis: Explore methods to detect circulating HIST1H2AG or its modifications in patient blood samples as potential biomarkers.

• Single-cell analysis: Employ single-cell Western blotting or mass cytometry with HIST1H2AG (Ab-74) antibody to capture expression heterogeneity within tumors.

• In situ hybridization correlation: Correlate protein expression (via immunohistochemistry) with mRNA levels (via in situ hybridization) to identify post-transcriptional regulation.

These methodologies provide complementary insights into the complex relationship between HIST1H2AG alterations and cancer development .

What role does HIST1H2AG play in viral infections and immune response?

While HIST1H2AG's specific role in viral infections is not directly addressed in the search results, research on H2A-reactive antibodies suggests potential relevance to viral immunity, particularly regarding HIV-1:

• Neutralization assays: Investigate whether antibodies generated against HIST1H2AG have virus-neutralizing capabilities, similar to H2A-reactive antibodies that can neutralize HIV-1 .

• Molecular mimicry analysis: Examine sequence and structural similarities between HIST1H2AG and viral proteins to identify potential molecular mimicry.

• TLR-mediated activation studies: Investigate how viral TLR ligands affect activation of HIST1H2AG-reactive B cells, similar to studies showing TLR stimulation can break tolerance to H2A .

• Infection models: Study changes in HIST1H2AG expression, localization, and modifications during viral infection using the Ab-74 antibody.

• Chromatin immunoprecipitation sequencing (ChIP-seq): Map HIST1H2AG occupancy changes during viral infection to identify potential roles in regulating antiviral gene expression.

• Auto-reactivity studies: Examine whether viral infections induce breaking of tolerance to HIST1H2AG, potentially contributing to post-infection autoimmunity.

These approaches can elucidate how histone variants like HIST1H2AG may participate in the complex interplay between viral infections and immune responses .

How does HIST1H2AG compare functionally with other H2A variants and what methodologies best capture these differences?

The H2A family includes numerous variants with distinct functions. To compare HIST1H2AG with other variants:

• Evolutionary analysis: Conduct phylogenetic analysis to understand the evolutionary relationships between HIST1H2AG and other H2A variants.

• Structural studies: Use structural biology approaches to compare the three-dimensional structure of nucleosomes containing different H2A variants.

• Genome-wide localization: Perform comparative ChIP-seq analysis of HIST1H2AG and other H2A variants (e.g., H2A.Z, H2A.X, macroH2A) to identify unique and overlapping genomic targets .

• Functional replacement studies: Conduct CRISPR-Cas9 mediated replacement of HIST1H2AG with other H2A variants to assess functional equivalence or divergence.

• Interaction proteomics: Compare protein interaction networks of different H2A variants using immunoprecipitation followed by mass spectrometry.

• Dynamics studies: Compare incorporation and turnover rates of different H2A variants using pulse-chase experiments with labeled histones.

These comparative approaches can reveal unique functional roles of HIST1H2AG within the diverse H2A variant family .

What emerging technologies will advance our understanding of HIST1H2AG function in chromatin regulation?

Emerging technologies that promise to advance our understanding of HIST1H2AG function include:

• CUT&RUN and CUT&Tag: These techniques offer higher resolution and lower background than traditional ChIP approaches for mapping histone variant localization.

• Proximity labeling: Technologies like BioID or APEX2 fused to HIST1H2AG can identify proximal proteins in living cells.

• Live-cell super-resolution microscopy: Techniques like PALM and STORM can visualize individual HIST1H2AG-containing nucleosomes in living cells.

• Single-molecule approaches: Optical tweezers and magnetic tweezers can measure how HIST1H2AG affects nucleosome stability and dynamics at the single-molecule level.

• Cryo-electron microscopy: Advanced cryo-EM techniques can resolve atomic-level structures of HIST1H2AG-containing nucleosomes and their complexes with chromatin remodelers.

• Multi-omics integration: Computational approaches integrating ChIP-seq, ATAC-seq, RNA-seq, and proteomics data can build predictive models of HIST1H2AG function in chromatin regulation.

These technologies will provide unprecedented insights into the molecular mechanisms by which HIST1H2AG contributes to chromatin structure and function.

Histone H2A Variant Comparison Table

Applications and Methods for HIST1H2AG (Ab-74) Antibody

HIST1H2AG (Ab-74) Antibody Specifications