HIST1H2AG (Ab-36) Antibody

What is HIST1H2AG and what biological functions does it serve?

HIST1H2AG is a histone H2A type 1 protein that belongs to the histone family, which are core components of nucleosomes - the basic unit of chromatin structure. Histones play crucial roles in DNA packaging, gene regulation, and chromatin remodeling through post-translational modifications. HIST1H2AG specifically contributes to chromatin structure and has been implicated in various nuclear processes including transcriptional regulation and DNA repair mechanisms . The protein is encoded by the H2AC11 gene (previously known as HIST1H2AG) and has a UniProt primary accession number of P0C0S8 . Notably, histone variants like H2A.B have been found to associate with RNA processing factors and RNA Polymerase II, suggesting potential roles in transcriptional regulation that may be relevant to HIST1H2AG function .

What applications can HIST1H2AG (Ab-36) Antibody be used for in research?

The HIST1H2AG (Ab-36) Antibody (catalog number orb416611) has been validated for multiple laboratory applications including:

• Chromatin Immunoprecipitation (ChIP)

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Immunofluorescence (IF)

• Immunohistochemistry (IHC)

• Western Blotting (WB)

This polyclonal antibody is raised in rabbit and shows reactivity with human samples . When designing experiments, researchers should consider the recommended dilution ranges specific to each application. Similar antibodies targeting HIST1H2AG may have different validated applications; for example, other variants have also been validated for immunoprecipitation (IP) or immunocytochemistry (ICC) .

What are the optimal storage conditions for maintaining HIST1H2AG antibody activity?

For optimal preservation of antibody activity, HIST1H2AG antibodies should be stored at -20°C for long-term storage . For short-term storage and frequent use (up to one month), storing at 4°C is acceptable . To prevent protein degradation from repeated freeze-thaw cycles, it is recommended to aliquot the antibody solution before freezing . The antibody is typically supplied in a buffer containing glycerol (ranging from 50% to 60%), along with stabilizing proteins such as BSA (0.5%) and preservatives like sodium azide (0.02%) or Proclin-300 (0.03%) . These components help maintain antibody structure and function during storage and thawing procedures.

How should I optimize the dilution of HIST1H2AG (Ab-36) Antibody for different applications?

Optimization of antibody dilution is critical for achieving specific signal with minimal background. Based on available data, the following starting dilution ranges are recommended for HIST1H2AG antibodies:

The optimal dilution should be determined empirically for each specific experimental setup, sample type, and detection method. It is advisable to perform a dilution series in preliminary experiments using relevant positive and negative controls . For Western blotting, validation data shows detection of HIST1H2AG in HeLa cell lysates, which can serve as a positive control .

What controls should be included when using HIST1H2AG antibodies in immunohistochemistry or immunofluorescence?

For rigorous experimental design with HIST1H2AG antibodies in imaging applications, the following controls should be incorporated:

• Positive tissue controls: Human testis tissue has shown positive staining for HIST1H2AG and can serve as a reliable positive control . HeLa cells have also demonstrated positive staining in immunofluorescence applications .

• Negative controls: Include samples known not to express the target protein, or use isotype control antibodies.

• Blocking peptide controls: When available, including a competition assay with the immunizing peptide can demonstrate specificity. For the HIST1H2AG (Ab-36) antibody, the immunogen is derived from a specific human HIST1H2AG sequence .

• Secondary antibody-only controls: To assess non-specific binding of the detection system.

• Tissue-specific expression controls: Validate staining patterns against known expression patterns from literature or databases.

Immunohistochemical staining has been validated on human testis tissue and lung cancer samples, while immunofluorescence analysis has been performed on HeLa cells . These can serve as reference points for expected staining patterns.

How can I verify the specificity of HIST1H2AG (Ab-36) Antibody in my experimental system?

Verifying antibody specificity is crucial for generating reliable data. For HIST1H2AG (Ab-36) Antibody, consider these approaches:

• Western blot analysis: Look for a single band at the expected molecular weight (approximately 14 kDa) . Western blot analysis has been performed on HL-60 whole cell lysates and HeLa cells to demonstrate specificity .

• Immunoprecipitation validation: Immunoprecipitation analysis using HeLa cells has been conducted to verify the antibody's ability to specifically pull down the target protein .

• Genetic knockdown/knockout controls: Compare staining between wild-type samples and those with reduced or eliminated expression of HIST1H2AG.

• Cross-reactivity assessment: Test the antibody on samples from different species to confirm the specified species reactivity. The HIST1H2AG (Ab-36) antibody is reported to be reactive with human samples only , while other HIST1H2AG antibodies may show reactivity with mouse and rat samples as well .

• Blocking peptide competition: Pre-incubation of the antibody with the immunizing peptide should abolish specific staining if the antibody is truly specific.

How can HIST1H2AG antibodies be utilized in chromatin immunoprecipitation (ChIP) studies?

ChIP is a powerful technique for studying protein-DNA interactions, and HIST1H2AG (Ab-36) Antibody has been validated for this application . When implementing ChIP with this antibody:

• Cross-linking optimization: Since HIST1H2AG is a histone protein tightly associated with DNA, standard formaldehyde cross-linking protocols (1% formaldehyde for 10 minutes at room temperature) are typically effective.

• Sonication parameters: Optimize sonication conditions to achieve chromatin fragments of 200-500 bp.

• Antibody concentration: Starting with 2-5 μg of antibody per ChIP reaction is recommended, with subsequent optimization.

• Controls: Include IgG control, input samples, and positive control antibodies (such as anti-H3).

• Analysis methods: Both qPCR and sequencing (ChIP-seq) can be used for downstream analysis of immunoprecipitated DNA.

High-throughput analysis has previously identified HIST1H2AG as potentially associated with promoter occupancy, suggesting it may play a role in transcriptional regulation . ChIP studies using HIST1H2AG antibodies can help elucidate its genomic distribution and potential regulatory functions.

What is known about the interaction between HIST1H2AG and RNA processing factors?

While specific information about HIST1H2AG interactions with RNA processing factors is limited in the provided search results, related histone variants provide valuable insights that may be relevant to HIST1H2AG research:

The histone variant H2A.B (a related H2A variant) has been found to associate with RNA processing factors and RNA Polymerase II in testis chromatin . Interestingly, many of these interactions (with factors like Sf3b155, Spt6, DDX39A, and RNA Pol II) were inhibited by the presence of endogenous RNA, suggesting RNA-dependent interactions . H2A.B has also been shown to bind RNA directly.

Researchers investigating HIST1H2AG might consider:

• RNA immunoprecipitation assays: To determine if HIST1H2AG similarly interacts with RNA molecules.

• Co-immunoprecipitation studies: To identify protein interaction partners, particularly RNA processing factors.

• RNA-seq analysis: To examine the impact of HIST1H2AG depletion on RNA processing and splicing patterns.

• Comparative studies: Investigating functional overlap between HIST1H2AG and other histone variants like H2A.B in relation to RNA processing.

These approaches could help determine whether HIST1H2AG shares functional characteristics with H2A.B in terms of RNA interactions and processing.

How does HIST1H2AG expression vary across different tissues and cell types?

Understanding the tissue-specific expression patterns of HIST1H2AG is important for experimental design and interpretation. Based on the available information and related histone variant studies:

• Tissue expression patterns: While comprehensive expression data for HIST1H2AG is not fully detailed in the search results, immunohistochemical validation has been performed on human testis tissue and lung cancer samples , suggesting expression in these tissues.

• Cell line expression: HIST1H2AG protein has been detected in HeLa cells (cervical cancer cell line) and HL-60 cells (promyelocytic leukemia cell line) , making these suitable positive controls for antibody validation.

• Related histone variant expression: The histone variant H2A.B shows highest expression in testis followed by brain in mammals , which might provide clues about HIST1H2AG expression patterns.

• Experimental considerations: When studying HIST1H2AG in a new tissue or cell type, preliminary experiments to confirm expression levels are recommended before proceeding with functional studies.

• Pathological relevance: The validation of HIST1H2AG antibodies in lung cancer samples suggests potential research applications in cancer biology, though specific alterations in expression or function in disease states require further investigation.

What are common issues when using HIST1H2AG antibodies in Western blotting and how can they be resolved?

When using HIST1H2AG antibodies for Western blotting, researchers may encounter several challenges:

• Multiple bands or unexpected molecular weight: HIST1H2AG has a calculated molecular weight of approximately 14 kDa . If multiple bands appear:

  • Check for post-translational modifications like acetylation or methylation

  • Verify sample preparation (complete denaturation and reduction)

  • Optimize antibody dilution (start with 1:500-1:2000)

  • Consider using different blocking agents to reduce non-specific binding

• Weak or no signal:

  • Increase protein loading (20-50 μg total protein per lane)

  • Reduce antibody dilution

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

  • Ensure transfer efficiency by staining membrane with Ponceau S

  • Use enhanced chemiluminescence detection systems

• High background:

  • Increase washing duration and frequency

  • Optimize blocking conditions (try 5% non-fat milk vs. BSA)

  • Increase antibody dilution

  • Ensure fresh buffers are used

• Sample preparation considerations:

  • For histone proteins, specialized extraction protocols may be necessary

  • Use histone extraction buffers containing high salt concentration

  • Include protease inhibitors and phosphatase inhibitors if studying modified histones

How can I differentiate between HIST1H2AG and other highly similar histone variants in my experiments?

Distinguishing between highly similar histone variants presents a significant challenge in histone biology research:

• Antibody selection: Choose antibodies raised against unique peptide sequences. The HIST1H2AG (Ab-36) Antibody is generated against a specific region of the human HIST1H2AG protein , which may provide better specificity compared to antibodies targeting conserved regions.

• Complementary techniques:

  • Mass spectrometry can distinguish variant-specific peptides

  • RNA interference targeting variant-specific sequences can help validate antibody specificity

  • Recombinant expression of tagged variants can be used as controls

• Bioinformatic analysis: When analyzing sequencing data:

  • Focus on variant-specific sequences rather than conserved regions

  • Use variant-specific primers for qPCR validation

  • Consider the genomic context and regulatory elements specific to each variant

• Functional assays: Different histone variants may have distinct functions:

  • H2A.B has been associated with RNA processing and RNA Polymerase II

  • Compare chromatin association patterns and genomic locations

  • Assess tissue-specific expression patterns (e.g., H2A.B is highly expressed in testis and brain)

• Cross-reactivity testing: Test the antibody against recombinant proteins of similar histone variants to assess potential cross-reactivity.

What approaches can be used to study post-translational modifications of HIST1H2AG?

Histone post-translational modifications (PTMs) are crucial for chromatin regulation and function. To study HIST1H2AG PTMs:

• Modification-specific antibodies: Use antibodies specific to particular modifications, such as:

  • Acetyl-HIST1H2AG (K13) Antibody for studying lysine 13 acetylation

  • Phosphorylation-specific antibodies targeting known or predicted modification sites

• Mass spectrometry approaches:

  • Top-down MS analysis of intact histones

  • Bottom-up MS analysis of histone peptides after enzymatic digestion

  • Middle-down MS approaches with limited proteolysis

  • Targeted MS methods for specific modifications

• Sequential ChIP (Re-ChIP):

  • First immunoprecipitate with anti-HIST1H2AG antibody

  • Follow with modification-specific antibodies

  • This approach reveals subpopulations of modified HIST1H2AG at specific genomic loci

• Functional correlation studies:

  • Compare PTM patterns with transcriptional activity

  • Analyze colocalization with transcription factors or chromatin modifiers

  • Study the dynamics of modifications during cellular processes like cell cycle, differentiation, or stress response

• Inhibitor studies: Use specific inhibitors of histone-modifying enzymes (e.g., HDAC inhibitors, HAT inhibitors) to manipulate modification levels and assess functional consequences.

How might HIST1H2AG be involved in pathological conditions or disease states?

While direct evidence linking HIST1H2AG to specific diseases is limited in the provided search results, several research directions are promising:

• Cancer biology: The validation of HIST1H2AG antibodies in lung cancer samples suggests potential roles in oncogenesis or tumor progression. Researchers might investigate:

  • Altered expression patterns in different cancer types

  • Correlation with clinical outcomes or treatment response

  • Potential epigenetic dysregulation involving HIST1H2AG in cancer

• Neurodevelopmental disorders: Related histone variants like H2A.B show expression in brain tissue , and histones generally play crucial roles in neurodevelopment. HIST1H2AG has been mentioned in the context of research related to disorders involving linguistic processing deficits , suggesting potential neurological relevance.

• Reproductive biology: Given the expression in testis tissue and the high expression of related variant H2A.B in testis , HIST1H2AG may have functions in spermatogenesis or reproductive processes.

• Epigenetic regulation: As a histone protein, HIST1H2AG likely contributes to epigenetic regulation, with potential implications for developmental disorders, aging-related conditions, and environmental response mechanisms.

• Inflammatory and immune responses: Histones can function as damage-associated molecular patterns (DAMPs) when released extracellularly. Investigation into whether HIST1H2AG contributes to inflammatory processes could be valuable.

What are the current methodological advances for studying histone variant dynamics in living cells?

Recent technological advances have expanded the toolkit for studying histone dynamics in living systems:

• Live-cell imaging approaches:

  • FRAP (Fluorescence Recovery After Photobleaching) to study exchange rates

  • Single-molecule tracking of fluorescently tagged histone variants

  • FRET-based sensors to detect conformational changes or interactions

• Engineered systems for controlled expression:

  • Inducible expression systems for temporal control

  • CRISPR/Cas9-mediated tagging of endogenous HIST1H2AG

  • Rapid protein degradation systems (e.g., auxin-inducible degron) for acute depletion

• Proximity labeling approaches:

  • BioID or TurboID fusion proteins to identify proximal interactors

  • APEX2-based proximity labeling for temporal resolution of the interactome

  • These methods can identify transient or context-specific interactions

• Chromatin capture technologies:

  • Advances in Hi-C and related methods to study 3D chromatin organization

  • CUT&RUN or CUT&Tag for high-resolution mapping with lower cell numbers

  • Single-cell approaches to study heterogeneity in histone variant distribution

• Genomic integration site mapping:

  • ChIP-seq with spike-in normalization for quantitative comparisons

  • CUT&RUN for higher signal-to-noise ratio in variant mapping

  • Integration with transcriptomic data to correlate with gene expression

These methodologies can be applied to study HIST1H2AG dynamics, providing insights into its functional roles in different cellular contexts and physiological states.