HIST1H3A (Ab-42) Antibody

What is HIST1H3A (Ab-42) Antibody and what epitope does it recognize?

HIST1H3A (Ab-42) Antibody is a rabbit polyclonal antibody that specifically recognizes the peptide sequence around the Arginine 42 (Arg42) site of human Histone H3.1. Histones are small, highly basic proteins consisting of a globular domain with unstructured N- and C-terminal tails protruding from the main structure . The antibody (product code CSB-PA010418OA42nme2HU) targets a specific region of Histone H3.1, which is encoded by the HIST1H3A gene and has multiple aliases including H3/a, H3/b, H3/c, and others .

The epitope around Arg42 is located within the core globular domain of the histone protein, which forms part of the nucleosome structure. This region is significant as it contributes to the histone-DNA interface and plays a role in nucleosome stability. The antibody is produced through antigen affinity purification and has been validated for use in ELISA and immunohistochemistry applications with human samples .

How does HIST1H3A (Ab-42) Antibody differ from other histone H3 antibodies?

HIST1H3A (Ab-42) Antibody differs from other histone H3 antibodies primarily in its epitope specificity. While many histone H3 antibodies target either total histone H3 protein (pan-H3 antibodies) or specific post-translational modifications, the HIST1H3A (Ab-42) Antibody specifically recognizes the region around Arginine 42 of the Histone H3.1 variant .

This distinguishes it from:

• Pan-histone H3 antibodies that detect endogenous levels of total histone H3 protein regardless of modifications or variants .

• Modification-specific antibodies like H3K4me3 antibody that recognize histone H3 with particular post-translational modifications, such as trimethylation at lysine 4 .

• Antibodies targeting different regions of the histone protein, such as the N-terminal tail where many post-translational modifications occur.

The specificity of histone antibodies varies considerably, as demonstrated by studies analyzing commercial histone tail PTM antibodies . The specificity factor (SF) ratio, which compares binding at the target site versus non-target sites, can range from as low as 5 to as high as 179 for different histone antibodies . The HIST1H3A (Ab-42) Antibody's focus on the Arg42 region provides researchers with a tool to specifically study this region of histone H3.1, which is particularly valuable when investigating variant-specific functions or structural roles.

What is the significance of targeting the Arg (42) site on Histone H3.1?

Targeting the Arg (42) site on Histone H3.1 offers several significant advantages for histone research:

• Structural relevance: Arg42 is located within the globular domain of histone H3.1, which is critical for nucleosome structure and stability. This region contributes to the histone-DNA interface within the nucleosome, making it important for chromatin organization.

• Potential for modification: Arginine residues in histones can undergo various post-translational modifications, including methylation and citrullination. These modifications can alter chromatin structure and function, affecting gene expression patterns.

• Variant specificity: Targeting this site may allow researchers to distinguish between different histone H3 variants, as there can be sequence variations or differential modification patterns at this position between variants.

• Chromatin regulation: Histone H3 plays a key role in regulating gene expression through modifications. Dysregulation of histone H3 has been implicated in various diseases, including cancer and neurological disorders .

• Protein-protein interactions: The globular domain of histones participates in interactions with chromatin remodeling complexes and other regulatory proteins. Antibodies targeting this region can help identify and study these interactions.

Histone H3.1 is encoded by the HIST1H3A gene (Gene ID: 8350) , and understanding specific regions of this protein, including the Arg42 site, contributes to our knowledge of epigenetic regulation and its role in normal development and disease pathology.

What are the validated applications for HIST1H3A (Ab-42) Antibody?

The HIST1H3A (Ab-42) Antibody has been validated for specific applications according to the manufacturer's data. Understanding these validated applications is essential for designing appropriate experiments:

While these are the specifically validated applications for the HIST1H3A (Ab-42) Antibody, it's informative to consider the broader range of applications for which histone H3 antibodies are commonly used:

For the HIST1H3A (Ab-42) Antibody specifically, researchers should begin with the validated applications and recommended dilutions. If exploring additional applications, thorough validation studies should be conducted, including appropriate positive and negative controls, to ensure reliable and reproducible results.

What is the recommended protocol for using HIST1H3A (Ab-42) Antibody in immunohistochemistry?

Based on the manufacturer's validated protocol , the following methodology is recommended for immunohistochemistry with HIST1H3A (Ab-42) Antibody:

Detailed IHC Protocol:

• Sample Preparation:

  • Use paraffin-embedded tissue sections

  • Perform dewaxing with xylene or suitable substitute

  • Rehydrate through a graded alcohol series to water

• Antigen Retrieval:

  • Method: High pressure in citrate buffer (pH 6.0)

  • This step is crucial for exposing epitopes masked during fixation

  • Duration: Typically 10-20 minutes (optimize based on tissue type)

• Blocking:

  • Block with 10% normal goat serum

  • Duration: 30 minutes

  • Temperature: Room temperature

  • Purpose: Reduces non-specific binding

• Primary Antibody Incubation:

  • Dilution: 1:20-1:200 (optimize for specific tissue type)

  • Diluent: 1% BSA in PBS

  • Incubation: Overnight at 4°C

  • Volume: Sufficient to cover tissue section completely

• Washing:

  • Buffer: PBS or TBS with 0.05% Tween-20

  • Duration: 3 × 5 minutes

  • Purpose: Removes unbound primary antibody

• Secondary Antibody:

  • Use a biotinylated secondary antibody appropriate for rabbit primary

  • Dilution: According to manufacturer's recommendation

  • Incubation: Typically 30-60 minutes at room temperature

• Detection System:

  • Visualize using an HRP conjugated SP system

  • Develop with DAB or other suitable chromogen

  • Counterstain with hematoxylin for nuclear definition

  • Mount with appropriate mounting medium

The protocol has been successfully demonstrated on human small intestine tissue . For optimal results, researchers should include appropriate controls and may need to adjust parameters based on their specific tissue type and experimental conditions.

How should HIST1H3A (Ab-42) Antibody be stored and handled to maintain activity?

Proper storage and handling of the HIST1H3A (Ab-42) Antibody is critical for maintaining its specificity and sensitivity. Based on the manufacturer's recommendations , the following guidelines should be followed:

Storage Conditions:

• Upon receipt, store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles as these can significantly degrade antibody quality

Storage Buffer Composition:

• 50% Glycerol

• 0.01M PBS, pH 7.4

• 0.03% Proclin 300 (preservative)

This buffer formulation helps maintain antibody stability during storage.

Recommended Handling Practices:

• Aliquoting:

  • Upon receipt, divide the antibody into small single-use aliquots

  • Use sterile microcentrifuge tubes

  • Record aliquot volumes and dates on each tube

  • This practice minimizes freeze-thaw cycles that can denature the antibody

• Thawing:

  • Thaw frozen aliquots at 4°C or on ice rather than at room temperature

  • Avoid rapid temperature changes that can cause protein denaturation

  • Centrifuge briefly before opening to collect all liquid at the bottom

• Working Solutions:

  • Prepare fresh working dilutions on the day of use

  • Do not store diluted antibody for extended periods

  • Return any unused stock solution to -20°C promptly after use

• Contamination Prevention:

  • Use sterile technique when handling the antibody

  • Use clean, nuclease-free pipette tips

  • Change tips between each handling step

• Documentation:

  • Maintain a log of antibody usage, including dates, applications, and observed results

  • Record lot numbers to track potential lot-to-lot variations

Following these storage and handling recommendations will help ensure consistent antibody performance across experiments and maximize the usable lifetime of the HIST1H3A (Ab-42) Antibody.

What controls should be used when working with HIST1H3A (Ab-42) Antibody?

Implementing appropriate controls is essential for validating results and ensuring experimental rigor when working with HIST1H3A (Ab-42) Antibody. Based on standard scientific practices and the available information, the following controls should be considered:

Essential Positive Controls:

• Validated Tissue Samples: Human small intestine tissue has been validated for this antibody in IHC applications . This or other tissues with known expression of HIST1H3A should be included as positive controls.

• Cell Lines with Confirmed Expression: HeLa cells are commonly used for histone H3 antibody validation . Other cell lines including LNCaP, HEK-293, Jurkat, and K-562 have also been used successfully with histone H3 antibodies .

• Recombinant Protein Controls: When available, purified recombinant HIST1H3A protein or synthetic peptides containing the target epitope can serve as positive controls in applications like Western blot or ELISA.

Critical Negative Controls:

• Primary Antibody Omission: Process identical samples with all reagents except the primary antibody, which should be replaced with antibody diluent only. This control identifies background from the secondary detection system.

• Isotype Control: Use a non-specific rabbit IgG at the same concentration as the primary antibody to assess non-specific binding due to the antibody class rather than epitope specificity.

• Blocking Peptide Competition: If available, pre-incubate the antibody with excess immunizing peptide to block specific binding sites. Loss of signal confirms antibody specificity.

• Low-Expression Tissue/Cells: Include samples known to express very low levels of the target protein to establish detection thresholds.

Methodological Controls:

• Antibody Titration: Test multiple dilutions to determine the optimal concentration that provides maximum specific signal with minimal background (recommended range: 1:20-1:200 for IHC) .

• Antigen Retrieval Optimization: Compare different antigen retrieval methods to ensure optimal epitope exposure without creating artifacts.

• Multi-Application Validation: When possible, verify findings across different techniques (e.g., if using IHC, confirm with Western blot if the antibody is validated for both).

Implementing these controls systematically will help ensure reliable and reproducible results when using the HIST1H3A (Ab-42) Antibody and facilitate troubleshooting if unexpected results occur.

How can specificity of HIST1H3A (Ab-42) Antibody be verified in experiments?

Verifying the specificity of HIST1H3A (Ab-42) Antibody is crucial for ensuring reliable experimental results. Based on standard practices in antibody validation and the available information about histone antibodies , the following methodological approaches are recommended:

Fundamental Verification Methods:

• Peptide Competition/Blocking Assay:

  • Pre-incubate the antibody with excess immunizing peptide (peptide sequence around Arg42 of human Histone H3.1)

  • Run parallel experiments with blocked and unblocked antibody

  • Specific signals should be significantly reduced or eliminated in the blocked condition

  • This is one of the most straightforward methods to confirm epitope specificity

• Genetic Models:

  • Use HIST1H3A knockdown or knockout models if available

  • Compare antibody reactivity between wild-type and knockdown/knockout samples

  • Specific signals should be reduced proportionally to knockdown efficiency

  • This method provides strong evidence of antibody specificity at the whole protein level

• Cross-Reactivity Analysis:

  • Test the antibody against related histone variants or proteins with similar epitopes

  • Calculate specificity factors similar to those described in the literature

  • A high specificity factor (SF) ratio indicates good discrimination between target and non-target sites

  • This is particularly important for histone antibodies due to high sequence conservation

Advanced Verification Approaches:

• Multiple Detection Methods:

  • Verify findings using different detection techniques (e.g., if using IHC, confirm with Western blot if applicable)

  • Consistent results across methodologically distinct techniques increase confidence in antibody specificity

  • Each method has different sample preparation conditions that can affect epitope accessibility

• Immunoprecipitation-Mass Spectrometry:

  • Perform IP with the antibody followed by mass spectrometry analysis

  • This identifies all proteins pulled down by the antibody

  • Confirms the presence of HIST1H3A and reveals any cross-reactive proteins

  • Particularly valuable for complex samples or when cross-reactivity is suspected

• Epitope Mapping:

  • Test antibody reactivity against a series of overlapping peptides covering the putative epitope region

  • This precisely defines the amino acid sequence recognized by the antibody

  • Helps predict potential cross-reactivity with similar sequences in other proteins

Implementing a combination of these verification methods provides robust evidence of antibody specificity and increases confidence in experimental results. For histone antibodies specifically, careful validation is essential due to the high sequence conservation among histone variants and the impact of post-translational modifications on epitope recognition .

What are common sources of background or non-specific binding with HIST1H3A (Ab-42) Antibody, and how can they be minimized?

When working with HIST1H3A (Ab-42) Antibody, researchers may encounter several sources of background or non-specific binding that can complicate data interpretation. Understanding these issues and implementing appropriate mitigation strategies is essential for generating clean, interpretable results:

Common Background Sources and Solutions:

• Insufficient Blocking:

  • Problem: Inadequate blocking allows non-specific binding to tissue proteins

  • Solution: Use fresh, high-quality blocking reagents; the validated protocol recommends 10% normal goat serum for 30 minutes at room temperature

  • Advanced approach: Consider incorporating 0.1-0.3% Triton X-100 in the blocking solution to improve penetration for complex tissues

• Cross-Reactivity with Similar Epitopes:

  • Problem: Histone proteins share high sequence similarity

  • Solution: Perform specificity validation as detailed in Question 8

  • Advanced approach: Pre-adsorb the antibody against related histone proteins if cross-reactivity is detected

• Endogenous Peroxidase Activity (in IHC):

  • Problem: Tissues contain endogenous peroxidases that can react with HRP detection systems

  • Solution: Include a peroxidase quenching step (e.g., 3% H₂O₂ for 10 minutes) before antibody application

  • Advanced approach: Optimize quenching time based on tissue type to minimize epitope damage

• Inappropriate Antibody Concentration:

  • Problem: Excessive antibody increases non-specific binding; too little reduces true signal

  • Solution: Perform systematic titration experiments; the recommended range for IHC is 1:20-1:200

  • Advanced approach: Create a titration curve for each new tissue type or application

Optimization Strategies:

• Diluent Optimization:

  • Add carrier proteins (e.g., 1% BSA as used in the validated protocol)

  • Include mild detergents (0.05-0.1% Tween-20) to reduce hydrophobic interactions

  • Consider adding 5% normal serum from the same species as the sample to reduce non-specific binding

• Washing Protocol Enhancement:

  • Increase number of washes (5-6 washes instead of standard 3)

  • Extend wash duration (10 minutes per wash)

  • Use gentle agitation during washing to improve removal of unbound antibody

  • Include 0.1% Tween-20 in wash buffers to reduce non-specific hydrophobic interactions

• Antigen Retrieval Optimization:

  • The validated protocol uses high pressure in citrate buffer (pH 6.0)

  • If background persists, test alternative methods:

    • EDTA buffer (pH 8.0) can provide different epitope exposure profiles

    • Enzymatic retrieval (e.g., proteinase K) may be gentler for some applications

  • Optimize retrieval time to balance epitope exposure and tissue integrity

• Detection System Refinement:

  • Use detection systems with amplification capabilities for weak signals

  • Consider polymer-based detection systems that can reduce background compared to biotin-based systems

  • For fluorescent applications, use directly conjugated secondary antibodies to reduce background

Implementing these strategies systematically, starting with the validated protocol conditions and making incremental adjustments, will help minimize background and non-specific binding, resulting in cleaner, more interpretable data with the HIST1H3A (Ab-42) Antibody.

Can HIST1H3A (Ab-42) Antibody be used to study post-translational modifications near the Arg (42) site?

The HIST1H3A (Ab-42) Antibody recognizing the region around Arginine 42 of histone H3.1 presents both opportunities and challenges for studying post-translational modifications (PTMs) in this region. While not originally designed for PTM detection, this antibody can be leveraged in several sophisticated research approaches:

Experimental Strategies:

• Epitope Masking Studies:

  • Methodology: Compare antibody binding efficiency between native and chemically modified histones

  • Analysis: Reduced binding to modified samples indicates the presence of modifications that obscure the epitope

  • Advanced approach: Combine with mass spectrometry to identify specific modifications causing masking

• Dual Immunolabeling Approaches:

  • Methodology: Perform co-staining with HIST1H3A (Ab-42) Antibody and modification-specific antibodies

  • Analysis: Mutually exclusive staining patterns suggest modifications at or near Arg42

  • Advanced approach: Use proximity ligation assays (PLA) to quantify spatial relationships between the Arg42 epitope and specific modifications

• Sequential ChIP (ChIP-reChIP):

  • Methodology: If validated for ChIP, use in sequential immunoprecipitation with PTM-specific antibodies

  • Analysis: This identifies genomic regions where both the Arg42 epitope is accessible and specific modifications are present

  • Advanced approach: Combine with next-generation sequencing for genome-wide analysis

Relevant Post-Translational Modifications:

• Arginine Methylation:

  • Arg42 itself could potentially be methylated (mono- or di-methylation)

  • This would likely interfere with antibody binding

  • Functional consequence: Methylation might affect protein-DNA interactions within the nucleosome

• Arginine Citrullination:

  • Conversion of arginine to citrulline by protein arginine deiminases

  • Results in loss of positive charge, potentially disrupting nucleosome stability

  • The antibody would likely not recognize citrullinated Arg42

• Nearby Modifications:

  • Lysine acetylation or methylation on residues near Arg42

  • Phosphorylation of nearby serine/threonine residues

  • These could induce conformational changes affecting antibody accessibility

Methodological Considerations:

• Complementary Techniques:

  • Mass spectrometry remains the gold standard for definitive PTM identification

  • Use the antibody as part of an integrated approach rather than as a standalone PTM detector

  • Consider chemical or enzymatic treatments that specifically remove certain modifications to confirm their presence

• Interpretation Challenges:

  • Loss of antibody binding could indicate:

    • Direct modification of the epitope

    • Conformational changes due to nearby modifications

    • Protein-protein interactions obscuring the epitope

  • Additional experiments are needed to distinguish between these possibilities

By employing these sophisticated approaches, researchers can use the HIST1H3A (Ab-42) Antibody as part of a comprehensive strategy to investigate the post-translational modification landscape around the Arg42 region of histone H3.1, potentially revealing novel insights into chromatin regulation mechanisms.

How can HIST1H3A (Ab-42) Antibody be used in epigenetic research?

The HIST1H3A (Ab-42) Antibody can serve as a valuable tool in epigenetic research by enabling the study of histone H3.1 distribution and dynamics in chromatin. Given that histone H3 plays a central role in chromatin structure and gene regulation , this antibody can contribute to several advanced epigenetic research approaches:

Chromatin Structure and Dynamics Applications:

• Histone Variant Incorporation Studies:

  • Methodology: Use the antibody to track histone H3.1 incorporation during DNA replication

  • Research question: How does replication-dependent H3.1 deposition differ from replication-independent H3.3 incorporation?

  • Advanced approach: Combine with cell cycle markers to study stage-specific incorporation patterns

• Nucleosome Occupancy Mapping:

  • Methodology: If validated for ChIP, use to map genome-wide distribution of histone H3.1

  • Research question: How does H3.1 distribution correlate with transcriptionally active versus silent regions?

  • Advanced approach: Compare results with DNase-seq or ATAC-seq data to correlate with chromatin accessibility

• Chromatin Compaction Analysis:

  • Methodology: Use in immunofluorescence to visualize nuclear distribution in different cell states

  • Research question: How does cellular differentiation affect H3.1 distribution patterns?

  • Advanced approach: Combine with super-resolution microscopy for detailed spatial analysis

Disease-Focused Applications:

• Cancer Epigenetics:

  • Methodology: Compare H3.1 distribution in normal versus cancerous tissues using IHC

  • Research question: Are there alterations in histone H3.1 incorporation patterns in specific cancer types?

  • Advanced approach: Correlate findings with clinical outcomes to identify potential prognostic markers

• Neurodegenerative Disorders:

  • Methodology: Examine H3.1 distribution in brain tissue samples from disease models

  • Research question: Does aberrant histone deposition contribute to neurological disease pathology?

  • Advanced approach: Combine with transcriptomic analysis to correlate with gene expression changes

Technical Implementation Strategies:

• Optimized Immunohistochemistry:

  • The antibody is validated for IHC (1:20-1:200 dilution)

  • Use this approach to examine tissue-specific patterns of histone H3.1 expression

  • Apply in developmental studies to track changes during differentiation

• Chromatin Immunoprecipitation Adaptation:

  • While not specifically validated for ChIP, other histone H3 antibodies have been successfully used in this application

  • If adapting for ChIP, start with protocols established for other H3 antibodies

  • Incorporate appropriate controls to validate specificity in this application

• Multi-parameter Imaging:

  • Combine with antibodies against histone modifications or chromatin remodelers

  • Use confocal or super-resolution microscopy to analyze spatial relationships

  • Quantify colocalization with image analysis software

• Flow Cytometry Applications:

  • Adapt for intracellular staining to quantify H3.1 levels across cell populations

  • Combine with cell cycle markers to study cell cycle-dependent changes

  • Use in sorting applications to isolate cells with different H3.1 levels for further analysis

By implementing these approaches, researchers can leverage the HIST1H3A (Ab-42) Antibody to advance our understanding of histone H3.1's role in epigenetic regulation and its implications for development, cellular differentiation, and disease pathology.

What considerations are important when using HIST1H3A (Ab-42) Antibody in multi-parameter experiments?

Multi-parameter experiments combining HIST1H3A (Ab-42) Antibody with other detection reagents require careful planning to ensure successful outcomes. When designing these complex experiments, researchers should address several critical technical and experimental design considerations:

Antibody Compatibility Factors:

• Host Species Considerations:

  • The HIST1H3A (Ab-42) Antibody is raised in rabbit

  • For co-staining, pair with antibodies raised in different host species (mouse, goat, etc.)

  • If multiple rabbit antibodies must be used, consider:

    • Sequential staining with complete blocking between rounds

    • Directly conjugated primary antibodies

    • Fragment antibody labeling kits to create directly conjugated primaries

• Detection System Selection:

  • For immunofluorescence:

    • Choose fluorophores with minimal spectral overlap

    • Consider brightness differences when selecting fluorophore combinations

    • Account for tissue autofluorescence when selecting detection channels

  • For chromogenic detection:

    • Use distinctly colored substrates for clear differentiation

    • Consider sequential detection with blocking between rounds

Protocol Optimization Requirements:

• Epitope Retrieval Compatibility:

  • The validated protocol uses high pressure in citrate buffer (pH 6.0)

  • All targets in a multiplex panel must be retrievable under the same conditions

  • If targets require different retrieval methods:

    • Test compromise conditions that work acceptably for all targets

    • Consider sequential staining with different retrieval methods between rounds

• Antibody Dilution Refinement:

  • The recommended 1:20-1:200 dilution for IHC may need adjustment in multiplex settings

  • Perform titration of each antibody individually and in combination

  • Signal intensity may be affected by the presence of other antibodies or detection reagents

• Incubation Sequence Optimization:

  • Test both simultaneous and sequential incubation approaches

  • For sequential application, determine the optimal order (generally highest affinity last)

  • Include additional blocking steps between antibody applications

Quality Control Measures:

• Comprehensive Controls:

  • Single-stained controls for each antibody to assess individual performance

  • Controls with individual antibodies omitted to detect cross-reactivity

  • Isotype controls to identify non-specific binding

  • Absorption controls (pre-incubation with immunizing peptide) to verify specificity

• Bleed-Through Assessment:

  • Acquire single-stained samples on all detection channels

  • Quantify and correct for spectral overlap using appropriate algorithms

  • Consider linear unmixing for confocal or spectral imaging applications

Application-Specific Considerations:

• Co-Immunoprecipitation:

  • Determine optimal buffer conditions that preserve all target interactions

  • Consider mild cross-linking to stabilize transient interactions

  • Use appropriate controls to distinguish specific from non-specific binding

• Chromatin Studies:

  • For sequential ChIP applications, optimize elution conditions between rounds

  • Consider fragment size carefully when studying closely positioned epitopes

  • Include input controls at each step to calculate enrichment accurately

• High-Content Imaging:

  • Establish consistent thresholds for positive signal detection

  • Use automated image analysis algorithms to quantify colocalization

  • Implement machine learning approaches for unbiased pattern recognition

By addressing these considerations systematically, researchers can successfully incorporate HIST1H3A (Ab-42) Antibody into complex multi-parameter experimental designs, enabling more comprehensive analysis of histone H3.1 biology in relation to other cellular components and processes.