HIST1H2AG (Ab-119) Antibody

What is HIST1H2AG and what biological role does it play?

HIST1H2AG encodes Histone H2A type 1, a core component of nucleosomes that wraps and compacts DNA into chromatin. This compaction limits DNA accessibility to cellular machineries that require DNA as a template. Histones play central roles in transcription regulation, DNA repair, DNA replication, and chromosomal stability. The accessibility of DNA is regulated through complex post-translational modifications of histones (often referred to as the "histone code") and nucleosome remodeling processes .

Histone H2A is one of the four core histones (H2A, H2B, H3, and H4) that form the octameric nucleosome structure, around which approximately 146 base pairs of DNA are wrapped. HIST1H2AG specifically belongs to the replication-dependent class of histones whose expression peaks during S-phase of the cell cycle.

What distinguishes the HIST1H2AG (Ab-119) antibody from other histone antibodies?

The HIST1H2AG (Ab-119) antibody is a rabbit polyclonal antibody that specifically recognizes a peptide sequence around lysine 119 of human Histone H2A type 1 . This site specificity is particularly important because lysine 119 is a known site for ubiquitination, a post-translational modification involved in gene silencing and DNA damage response pathways .

Unlike some general H2A antibodies, the Ab-119 variant provides specificity for the region around this critical lysine residue, making it valuable for studies investigating specific post-translational modifications and their biological consequences .

What applications is the HIST1H2AG (Ab-119) antibody validated for?

According to product documentation, the HIST1H2AG (Ab-119) antibody has been validated for multiple applications:

The specific applications and recommended dilutions may vary slightly between manufacturers .

How should I store and handle the HIST1H2AG (Ab-119) antibody to maintain optimal activity?

Proper storage and handling are crucial for maintaining antibody performance:

• Upon receipt, store the antibody at -20°C or -80°C according to manufacturer recommendations .

• Avoid repeated freeze/thaw cycles as they can denature the antibody and reduce its efficacy .

• Consider preparing small aliquots for single use to minimize freeze/thaw cycles .

• The antibody is typically supplied in a buffer containing 50% glycerol and 0.01M PBS at pH 7.4 with 0.03-0.05% Proclin as a preservative .

• When working with the antibody, keep it on ice and return to storage promptly after use.

What controls should be included when using this antibody in experimental workflows?

Proper experimental controls are essential for validating results:

Positive Controls:

• Cell lines or tissues known to express HIST1H2AG (virtually all nucleated human cells)

• Recombinant HIST1H2AG protein for Western blot

• Samples with known ubiquitination at K119 when studying this modification

Negative Controls:

• Primary antibody omission control

• Isotype control (rabbit IgG)

• Blocking peptide competition assay using the immunizing peptide

• HIST1H2AG-depleted samples (via knockdown or knockout) if available

Loading and Processing Controls:

• Total protein stain for Western blots

• Housekeeping proteins as loading controls

• Non-specific IgG for ChIP experiments

What antigen retrieval methods work best for HIST1H2AG (Ab-119) in immunohistochemistry?

Effective antigen retrieval is critical for IHC success with histone antibodies like HIST1H2AG (Ab-119). Cross-linking fixatives like formalin can mask epitopes by creating protein-protein crosslinks that bury the target antigen .

Based on IHC troubleshooting guidance for histone antibodies, the following approaches are recommended:

• Heat-Induced Epitope Retrieval (HIER):

  • Citrate buffer (pH 6.0) at 95-100°C for 20 minutes

  • EDTA buffer (pH 8.0) at 95-100°C for 20 minutes

• Enzymatic Retrieval (less common for histones):

  • Proteinase K (20 μg/ml) for 10-15 minutes at room temperature

For optimal results, different retrieval methods should be empirically tested, as the accessibility of the lysine 119 region can be affected by fixation conditions and tissue type .

What are the most common causes of false negative results with HIST1H2AG (Ab-119) antibody?

False negative results can occur for several reasons:

• Ineffective Antigen Retrieval: The epitope may remain masked by fixation crosslinks .

  • Solution: Optimize antigen retrieval by testing different methods (heat vs. enzymatic) and buffer compositions.

• Excessive Fixation: Over-fixation can permanently mask epitopes.

  • Solution: Optimize fixation time and explore different fixatives if preparing samples in-house.

• Antibody Degradation: Improper storage or handling can diminish antibody activity.

  • Solution: Use fresh aliquots and verify antibody activity with positive controls.

• Post-translational Modifications: If the epitope site (Lys-119) is heavily modified (e.g., ubiquitinated), it might prevent antibody binding.

  • Solution: Consider using antibodies specific to the modified form or treatment with deubiquitinating enzymes if studying the unmodified protein.

• Low Expression Levels: Target protein might be expressed at levels below detection threshold.

  • Solution: Use more sensitive detection methods or signal amplification techniques.

What strategies can resolve high background issues when using this antibody in immunostaining?

High background is a common problem that can obscure specific signals:

• Optimize Antibody Concentration: Titrate the antibody to find the optimal working dilution.

  • The recommended dilution range for IHC is 1:10-1:100 or 5-20 μg/ml .

  • For IF/ICC, 5-20 μg/ml is recommended .

• Blocking Optimization:

  • Increase blocking time or concentration (try 5% BSA in TBST) .

  • Add 5-10% serum from the species of the secondary antibody to the blocking solution.

  • Consider specialized blocking reagents for difficult samples.

• Secondary Antibody Issues:

  • Ensure secondary antibody is appropriate for the host species (rabbit).

  • Reduce secondary antibody concentration if signal is too strong.

  • Include proper controls (secondary-only, isotype controls).

• Washing Steps:

  • Increase number and duration of washing steps.

  • Use gentle agitation during washes.

  • Ensure buffer composition is appropriate (typically PBS or TBS with 0.05-0.1% Tween-20).

• Tissue/Sample Preparation:

  • Ensure proper fixation and permeabilization.

  • Consider autofluorescence quenching for fluorescence applications.

  • Use fresh sections or samples when possible.

How can the HIST1H2AG (Ab-119) antibody be optimized for chromatin immunoprecipitation (ChIP) studies?

ChIP optimization with HIST1H2AG (Ab-119) requires careful consideration of several factors:

• Crosslinking Conditions:

  • For histone studies, standard formaldehyde crosslinking (1% for 10 minutes at room temperature) is typically sufficient.

  • For studying interactions with other proteins, consider dual crosslinking with both formaldehyde and protein-protein crosslinkers like DSG or EGS.

• Sonication Parameters:

  • Aim for chromatin fragments of 200-500 bp for standard ChIP.

  • Optimize sonication time, amplitude, and pulse settings for your specific sonicator model.

  • Verify fragment size by agarose gel electrophoresis before proceeding.

• Antibody Amount and Incubation:

  • Start with 2-5 μg of antibody per ChIP reaction.

  • Incubate overnight at 4°C with rotation to ensure maximal binding.

  • Include appropriate controls (input, IgG, positive control regions).

• Washing Stringency:

  • Balance between stringent washing to reduce background and preserving specific interactions.

  • Consider titrating salt concentration in wash buffers.

• Elution and Recovery:

  • Ensure complete reversal of crosslinking (typically 65°C for 4-6 hours or overnight).

  • Include RNase and Proteinase K treatments to ensure clean DNA recovery.

What considerations are important when studying HIST1H2AG post-translational modifications?

Studying histone post-translational modifications (PTMs) requires special attention to several factors:

• Epitope Specificity and Accessibility:

  • The HIST1H2AG (Ab-119) antibody targets the region around Lys-119, which is a known ubiquitination site .

  • Consider whether you need antibodies specific to the modified form (e.g., H2A ubiquityl K119) vs. the unmodified form.

• Preserving Modification Status:

  • Include deacetylase inhibitors (e.g., sodium butyrate, TSA) and phosphatase inhibitors in lysis buffers.

  • For ubiquitination studies, include deubiquitinase inhibitors like N-ethylmaleimide.

  • Process samples quickly and keep at cold temperatures to minimize enzymatic removal of modifications.

• Verification Approaches:

  • Use multiple antibodies targeting the same modification from different vendors.

  • Consider orthogonal approaches like mass spectrometry to confirm modifications.

  • Use cells/tissues with genetic or pharmacological manipulation of the enzymes responsible for the modification.

• Quantification Methods:

  • For Western blots, normalize modified histone signal to total histone levels.

  • For imaging, use appropriate controls and consistent acquisition parameters.

  • Consider the dynamic range of your detection method relative to the biological range of the modification.

How does HIST1H2AG (Ab-119) antibody contribute to understanding chromatin dynamics and epigenetic regulation?

The HIST1H2AG (Ab-119) antibody targeting the K119 region provides valuable insights into chromatin dynamics:

• Polycomb Repressive Complex Function:

  • H2A ubiquitination at K119 is catalyzed by the E3 ubiquitin ligase Ring1B, a component of Polycomb Repressive Complex 1 (PRC1).

  • This modification is associated with gene silencing and X-chromosome inactivation.

  • Studies using this antibody can help map the genome-wide distribution of this repressive mark.

• Chromatin Remodeling:

  • H2A modifications influence nucleosome stability and accessibility.

  • The antibody can be used to investigate how H2A status changes during processes requiring chromatin remodeling.

• Cell Differentiation and Development:

  • Histone modifications, including those on H2A, play crucial roles in cell fate decisions.

  • The antibody allows tracking of specific chromatin states during differentiation processes.

• DNA Damage Response:

  • H2A and its variants are modified during DNA damage response.

  • This antibody can help elucidate how the K119 region may be involved in DNA repair pathways.

• Disease Mechanisms:

  • Alterations in histone modifications are implicated in various diseases, particularly cancer.

  • The antibody provides a tool to investigate these changes in disease models and patient samples.

What are the optimal sample preparation protocols for Western blot using HIST1H2AG (Ab-119) antibody?

Optimal Western blot protocols for histone analysis require special considerations:

• Histone Extraction:

  • Acid extraction methods are preferred for enriching histones:

    • Treat cell pellets with 0.2M H₂SO₄ or 0.25M HCl for 30 minutes on ice

    • Precipitate histones with trichloroacetic acid or acetone

    • Resuspend in water or appropriate buffer

  • Commercial histone extraction kits are also available and recommended for consistent results.

• Gel and Transfer Parameters:

  • Use high percentage (15-18%) polyacrylamide gels to properly resolve the low molecular weight (approximately 14-15 kDa) histone proteins.

  • Consider specialized systems like Triton-Acid-Urea (TAU) gels for separating histone variants and modifications.

  • For transfer, use PVDF membranes (rather than nitrocellulose) and add 0.02% SDS to transfer buffer.

  • Transfer at lower voltage (30V) for longer time (2 hours or overnight) to ensure efficient transfer of small proteins.

• Blocking and Antibody Incubation:

  • Block with 5% BSA in TBST rather than milk (milk contains bioactive proteins that may interfere with histone antibody binding) .

  • Incubate with primary antibody (diluted 0.01-2 μg/ml) overnight at 4°C.

  • Wash thoroughly with TBST (at least 3x10 minutes).

• Detection Optimization:

  • Use high-sensitivity ECL substrates due to the relatively low abundance of specific histone variants.

  • Consider fluorescent secondary antibodies for more accurate quantification.

How should immunofluorescence experiments be designed for optimal HIST1H2AG detection?

Immunofluorescence with histone antibodies requires specific considerations:

• Fixation and Permeabilization:

  • 4% paraformaldehyde for 10-15 minutes at room temperature is standard.

  • For nuclear antigens like histones, thorough permeabilization is crucial (try 0.5% Triton X-100 for 10 minutes).

  • Some protocols benefit from pre-extraction with detergent before fixation to remove soluble proteins and reduce background.

• Blocking and Antibody Incubation:

  • Block with 5% BSA or 10% normal serum in PBS with 0.1% Triton X-100.

  • Use the antibody at the recommended concentration of 5-20 μg/ml .

  • Incubate overnight at 4°C in a humidified chamber to prevent drying.

• Controls and Counterstaining:

  • Include a DNA counterstain like DAPI or Hoechst to confirm nuclear localization.

  • Consider co-staining with other nuclear markers to establish subcellular context.

  • Include appropriate negative controls (primary antibody omission, isotype control).

• Image Acquisition:

  • Use appropriate filter sets to avoid bleed-through between channels.

  • Collect Z-stacks when possible to capture the full nuclear volume.

  • Set exposure times based on negative controls to avoid false positives.

• Quantification Approaches:

  • For intensity measurements, use consistent acquisition parameters across all samples.

  • Consider nuclear segmentation based on DNA counterstain for automated analysis.

  • For co-localization studies, apply appropriate statistical measures beyond visual assessment.