HIST1H2AG (Ab-9) Antibody

What is HIST1H2AG (Ab-9) Antibody and what does it target?

HIST1H2AG (Ab-9) Antibody is a rabbit polyclonal antibody that specifically targets Human Histone H2A type 1 (H2A.1) around the site of Lysine 9 (Lys 9). This antibody recognizes a specific epitope in this histone protein, which is a critical component of the nucleosome structure in chromatin. The "Ab-9" designation indicates that the antibody specifically targets the Lysine 9 site on the histone protein. According to the product information, the target protein has multiple synonyms including H2AC11, H2AFP, HIST1H2AG, H2AC13, H2AFC, HIST1H2AI, H2AC15, H2AFD, HIST1H2AK, H2AC16, H2AFI, HIST1H2AL, H2AC17, H2AFN, and HIST1H2AM. The UniProt accession number for the target protein is P0C0S8, which helps researchers access additional structural and functional information about this histone variant .

What validated applications have been established for this antibody?

HIST1H2AG (Ab-9) Antibody has been validated for multiple research applications through rigorous testing protocols. The antibody has demonstrated efficacy in:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blot (WB)

• Immunohistochemistry (IHC)

• Immunofluorescence (IF)

• Chromatin Immunoprecipitation (ChIP)

Supporting validation data includes Western blot analysis of 293 whole cell lysates, immunohistochemistry of paraffin-embedded human glioma tissue, immunofluorescence staining of HeLa cells, and chromatin immunoprecipitation with HeLa cells treated with Micrococcal Nuclease . These validations across multiple technical platforms provide researchers with confidence in the antibody's performance across different experimental conditions.

What are the optimal storage conditions for preserving antibody activity?

For maximum antibody stability and performance, HIST1H2AG (Ab-9) Antibody requires specific storage conditions:

The antibody is provided in a stabilizing buffer containing 0.03% Proclin 300 as a preservative and 50% Glycerol in 0.01M PBS (pH 7.4) as constituents . For optimal performance, it is highly recommended to aliquot the antibody into smaller volumes upon receipt to minimize freeze-thaw cycles, which can significantly degrade antibody activity and specificity. When properly stored, the antibody maintains its activity for approximately 12 months from the date of receipt .

What are the recommended dilution ranges for various experimental applications?

The optimal dilution of HIST1H2AG (Ab-9) Antibody varies significantly depending on the specific application and experimental conditions. Based on validation studies, the following dilution ranges have been established:

For ELISA and ChIP applications, researchers should perform titration experiments to determine optimal concentrations for their specific experimental systems . It is always advisable to begin with a mid-range dilution and adjust based on signal-to-noise ratio in preliminary experiments. The concentration of the antibody is lot-specific, and researchers should refer to the datasheet included with each specific lot for more precise guidance .

How should I design ChIP experiments using this antibody for epigenetic studies?

For effective chromatin immunoprecipitation (ChIP) using HIST1H2AG (Ab-9) Antibody, follow this validated protocol framework:

• Sample Preparation:

  • Culture approximately 4×10^6 cells (as validated with HeLa cells)

  • Crosslink with 1% formaldehyde for 10 minutes at room temperature

  • Quench with glycine (125mM final concentration)

  • Treat samples with Micrococcal Nuclease to fragment chromatin to 150-500 bp

  • Sonicate to solubilize chromatin and enhance fragmentation

• Immunoprecipitation:

  • Use 5μg of HIST1H2AG (Ab-9) Antibody per reaction

  • Include a normal rabbit IgG control for background assessment

  • Incubate overnight at 4°C with rotation

• Analysis:

  • Quantify the recovered ChIP DNA using real-time PCR

  • Use validated primers (such as those for β-Globin promoter region)

  • Calculate enrichment relative to input and IgG controls

This approach has been validated as shown in the ChIP experiment data where HeLa cells were successfully immunoprecipitated with the antibody followed by quantification using real-time PCR with primers against the β-Globin promoter region .

What controls are essential when using this antibody in immunohistochemistry?

When performing immunohistochemistry with HIST1H2AG (Ab-9) Antibody, implement these critical controls to ensure data validity:

• Positive Control Tissues:

  • Human glioma tissue sections (validated in product data)

  • Cell lines with known H2A expression (e.g., HeLa, 293 cells)

• Negative Controls:

  • Primary antibody omission (secondary antibody only)

  • Non-immune rabbit IgG at equivalent concentration

  • Peptide competition (pre-incubation with immunizing peptide)

• Procedural Controls:

  • Antigen retrieval optimization

  • Blocking optimization using 5% BSA or 10% normal serum

  • Antibody dilution series (1:20-200 as recommended)

• Analytical Controls:

  • Include regions of the section known to be negative

  • Assess nuclear vs. cytoplasmic staining patterns

  • Evaluate staining intensity variation across different cell types

Proper implementation of these controls will help distinguish specific binding from background or artifactual staining, particularly when quantitative analyses are planned . The validation data showing clear nuclear staining patterns in human glioma tissue provides a reference standard for expected results.

How can I optimize dual immunofluorescence protocols with HIST1H2AG (Ab-9) Antibody?

For successful dual immunofluorescence incorporating HIST1H2AG (Ab-9) Antibody, consider these methodological refinements:

• Sequential Antibody Application:

  • When using multiple rabbit-derived antibodies, implement sequential staining protocols

  • Apply HIST1H2AG (Ab-9) Antibody first at 1:50-200 dilution

  • Block with excess anti-rabbit Fab fragments before applying second primary antibody

• Fluorophore Selection:

  • Choose spectrally distinct fluorophores with minimal overlap

  • For nuclear co-localization studies, select fluorophores with different excitation/emission profiles

  • Consider brightness differentials when target proteins have significantly different expression levels

• Signal Amplification:

  • For low-abundance targets, implement tyramide signal amplification

  • For HIST1H2AG (Ab-9) Antibody, use biotin-streptavidin systems if direct detection yields insufficient signal

• Image Acquisition Parameters:

  • Collect sequential rather than simultaneous channel acquisition

  • Optimize exposure settings to prevent signal saturation

  • Implement appropriate bleed-through controls for each fluorophore combination

The successful implementation of immunofluorescence staining in HeLa cells with this antibody demonstrates its compatibility with fluorescence-based detection systems . When designing dual staining protocols, researchers should consider the nuclear localization of H2A and plan accordingly for co-localization studies with other nuclear proteins.

What methodological approaches can resolve inconsistent Western blot results with this antibody?

When troubleshooting variable Western blot results with HIST1H2AG (Ab-9) Antibody, implement this systematic approach:

• Sample Preparation Optimization:

  • Use specialized histone extraction methods (acid extraction with 0.2N HCl)

  • Include histone deacetylase inhibitors (sodium butyrate, trichostatin A)

  • Add phosphatase and protease inhibitor cocktails to all lysis buffers

• Gel Electrophoresis Refinements:

  • Use high percentage (15-18%) gels to resolve low molecular weight histones

  • Implement SDS-Triton-Acid-Urea (STAU) gels for separation of modified histones

  • Load appropriate amount of protein (10-20 μg for whole cell lysates)

• Transfer Parameter Adjustments:

  • Utilize PVDF membranes (0.2 μm pore size) for optimal histone retention

  • Implement extended transfer times at lower voltage

  • Consider specialized transfer buffers with reduced methanol for small proteins

• Antibody Incubation Optimization:

  • Test dilution range (1:500-5000) to identify optimal concentration

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

  • Implement additional blocking steps with 5% non-fat milk or BSA

• Detection System Calibration:

  • Compare chemiluminescent vs. fluorescent detection systems

  • Optimize exposure times to prevent signal saturation

  • Consider signal enhancement systems for low-abundance targets

The validation data showing successful detection in 293 whole cell lysates provides a reference point for expected band patterns (approximately 14 kDa) . Implementing these optimizations should help resolve inconsistent results and improve reproducibility across experiments.

How does this antibody perform in distinguishing between different histone H2A variants?

HIST1H2AG (Ab-9) Antibody's specificity for histone variants requires careful consideration:

• Variant Recognition Profile:

  • This antibody recognizes multiple H2A variants as indicated by the synonyms list

  • Target variants include HIST1H2AG, HIST1H2AI, HIST1H2AK, HIST1H2AL, and HIST1H2AM

  • All recognized variants belong to the Histone H2A type 1 family

• Distinguishing Related Variants:

  • The antibody does not distinguish between the highly similar type 1 H2A variants

  • For variant-specific detection, complementary approaches may be necessary

  • Mass spectrometry is recommended for definitive variant identification

• Modification Sensitivity:

  • Being targeted to the Lys9 region, modifications at this site may affect recognition

  • Acetylation, methylation, or other PTMs at Lys9 could potentially interfere with binding

  • Researchers studying specific modifications should validate recognition patterns

• Application-Specific Considerations:

  • For Western blot: The antibody may detect multiple bands corresponding to different variants

  • For ChIP: The antibody will immunoprecipitate chromatin containing any recognized H2A variant

  • For IF/IHC: The antibody will yield a composite signal of all recognized variants

When absolute variant specificity is required, researchers should consider using this antibody in combination with other techniques like mass spectrometry or variant-specific antibodies targeting unique regions .

What strategies can resolve high background in immunohistochemistry experiments?

For reducing non-specific background staining when using HIST1H2AG (Ab-9) Antibody in immunohistochemistry, implement this hierarchical approach:

• Blocking Protocol Enhancement:

  • Extend blocking time to 60 minutes at room temperature

  • Test alternative blocking agents (5% BSA, 10% normal goat serum)

  • Implement dual blocking with protein blockers and serum

  • Consider commercial blocking reagents designed for rabbit antibodies

• Antibody Dilution Optimization:

  • Begin with 1:100 dilution (mid-range of recommended 1:20-200)

  • Prepare a dilution series (1:50, 1:100, 1:200) to identify optimal concentration

  • Dilute antibody in blocking solution rather than buffer alone

• Washing Procedure Refinement:

  • Increase number of washes (minimum 3 × 5 minutes)

  • Use 0.1% Tween-20 in wash buffer to reduce hydrophobic interactions

  • Implement agitation during washing steps

  • Ensure thorough washing between all steps

• Tissue-Specific Optimizations:

  • For tissues with high endogenous peroxidase, extend H₂O₂ quenching (15-30 minutes)

  • For tissues with high biotin content, implement avidin-biotin blocking

  • For autofluorescent tissues, consider Sudan Black B treatment

The validation data showing clean nuclear staining in human glioma tissue demonstrates that appropriate optimization can yield specific signals with minimal background . Systematic implementation of these approaches should significantly improve signal-to-noise ratio in challenging samples.

How can I verify antibody specificity for critical research applications?

To rigorously validate HIST1H2AG (Ab-9) Antibody specificity for high-stakes research applications, implement this comprehensive validation strategy:

• Peptide Competition Assay:

  • Pre-incubate antibody with excess immunizing peptide (Lys9 region peptide)

  • Run parallel experiments with blocked and unblocked antibody

  • Specific signals should be eliminated or significantly reduced

• Genetic Validation:

  • Test antibody on samples with CRISPR/Cas9 knockout of target

  • Alternatively, use siRNA knockdown to create partially depleted samples

  • Compare signal intensity between wild-type and depleted samples

• Orthogonal Method Validation:

  • Confirm findings using alternative detection methods

  • Compare results with antibodies targeting different epitopes of the same protein

  • Correlate antibody-based detection with mRNA expression data

• Cross-Reactivity Assessment:

  • Test reactivity on recombinant proteins of closely related histone variants

  • Evaluate species cross-reactivity if working with non-human samples

  • Document any potential cross-reactive proteins

• Application-Specific Validation:

  • For ChIP: Perform ChIP-seq and analyze peak distribution patterns

  • For IF/IHC: Correlate staining patterns with known biological distributions

  • For WB: Confirm molecular weight and compare with predicted size

The antibody's performance across multiple applications (ELISA, WB, IHC, IF, ChIP) as documented in the validation data provides a foundation for these specificity assessments . For publication-quality research, implementing multiple validation approaches will strengthen data interpretation and reliability.

How can HIST1H2AG (Ab-9) Antibody contribute to chromatin dynamics research?

HIST1H2AG (Ab-9) Antibody offers several methodological approaches for investigating chromatin dynamics:

• Histone Exchange Studies:

  • Use ChIP-seq with this antibody to map genome-wide H2A distribution

  • Compare occupancy patterns before and after cellular stimulation

  • Identify regions with dynamic H2A incorporation or eviction

• Chromatin Remodeling Analysis:

  • Combine ChIP using this antibody with nucleosome positioning assays

  • Correlate H2A occupancy with nucleosome density and positioning

  • Investigate relationship between H2A variants and chromatin accessibility

• Cell Cycle Dynamics:

  • Perform synchronized cell experiments with IF staining

  • Track changes in H2A distribution throughout cell cycle phases

  • Correlate with cell cycle markers to identify phase-specific patterns

• DNA Damage Response:

  • Use dual IF to study co-localization with γH2AX after damage induction

  • Perform ChIP-seq before and after DNA damage to map recruitment patterns

  • Investigate H2A exchange at repair sites using time-course experiments

• Differential Nuclear Localization:

  • Implement super-resolution microscopy with this antibody

  • Map H2A distribution within nuclear compartments

  • Correlate localization with transcriptional activity markers

The antibody's validated performance in both ChIP and immunofluorescence applications makes it particularly suitable for studying dynamic chromatin processes . These approaches can provide insights into the role of H2A variants in regulating genome function and stability.

What considerations are important for quantitative chromatin immunoprecipitation experiments?

For rigorous quantitative ChIP experiments using HIST1H2AG (Ab-9) Antibody, implement these methodological controls:

• Experimental Design Considerations:

  • Include input DNA controls (typically 1-10% of starting material)

  • Use normal rabbit IgG as negative control at equivalent concentration

  • Include positive control regions (housekeeping gene promoters)

  • Design primers for regions without expected H2A enrichment as baseline

• Chromatin Preparation Standardization:

  • Standardize cell numbers (4×10^6 cells as used in validation experiments)

  • Verify chromatin fragmentation size (150-500 bp) by gel electrophoresis

  • Document crosslinking efficiency with pilot experiments

  • Standardize Micrococcal Nuclease digestion conditions

• Quantification Calibration:

  • Prepare standard curves for all qPCR primer sets

  • Calculate primer efficiencies and incorporate into analysis

  • Use multiple reference genes for normalization

  • Apply appropriate statistical methods for significance testing

• Technical Implementation:

  • Perform a minimum of three biological replicates

  • Include technical triplicates for each qPCR measurement

  • Use consistent threshold settings across all experiments

  • Document all normalization methods and calculations

The validation data showing successful ChIP with quantification against the β-Globin promoter provides a procedural foundation . Implementation of these quantitative controls will enhance reproducibility and statistical validity of ChIP experiments using this antibody.