HIST1H2AG (Ab-118) Antibody

What are the validated applications for HIST1H2AG (Ab-118) Antibody?

The HIST1H2AG (Ab-118) Polyclonal Antibody has been validated for multiple research applications including ELISA, Western Blot (WB), Immunoprecipitation (IP), Chromatin Immunoprecipitation (ChIP), Immunohistochemistry (IHC), and Immunofluorescence (IF). These applications provide researchers with versatile options for detecting histone H2A type 1 in various experimental contexts . The antibody's broad application profile makes it suitable for both protein quantification and localization studies, with particular strength in chromatin-based research applications.

What are the recommended dilution ranges for different applications?

For optimal results, researchers should use the following application-specific dilution ranges:

• Western Blot: 1:500-5000

• Immunohistochemistry (paraffin-embedded sections): 1:20-200

• Immunofluorescence: 1:50-200

These ranges should be considered starting points for optimization, as actual optimal dilutions may vary depending on sample type, preparation method, and detection system employed. Preliminary titration experiments are recommended when working with new sample types.

What species reactivity has been confirmed for this antibody?

The HIST1H2AG (Ab-118) Antibody has confirmed reactivity with human (Homo sapiens) samples across multiple applications . Some product variants also demonstrate reactivity with mouse (Mus musculus) samples . The cross-species reactivity reflects the highly conserved nature of histone proteins across mammalian species, though researchers should verify reactivity when working with species not explicitly listed in the product specifications.

What is the immunogen used to generate this antibody?

This polyclonal antibody was generated using a peptide sequence around the site of Lysine 118 derived from Human Histone H2A type 1 . The specific immunogen design targeting this region allows for detection of HIST1H2AG protein in its native and denatured forms, making it suitable for both structural and functional studies of histone H2A type 1.

What are the proper storage conditions for maintaining antibody activity?

To maintain optimal activity, the HIST1H2AG (Ab-118) Antibody should be stored refrigerated at 2-8°C for short-term use (up to 2 weeks). For long-term storage, it should be kept at -20°C in small aliquots to prevent freeze-thaw cycles that could degrade the antibody . The antibody is supplied in a buffer containing 50% glycerol and 0.03% Proclin 300 as a preservative, which helps maintain stability during storage .

How can the HIST1H2AG (Ab-118) Antibody be utilized in epigenetic research?

This antibody serves as a valuable tool in epigenetic research through its validated use in Chromatin Immunoprecipitation (ChIP) assays . Researchers can employ it to investigate histone H2A distribution patterns across the genome, histone modifications associated with specific genomic regions, and changes in chromatin structure during cellular processes like differentiation or disease progression. When combined with sequencing (ChIP-seq) or qPCR techniques, this antibody enables genome-wide mapping of H2A-associated regulatory elements and their dynamics during biological processes.

What considerations should be taken when designing experiments involving both HIST1H2AG detection and other histone modification studies?

When designing multi-parameter histone studies, researchers should consider potential epitope masking effects where histone modifications may interfere with antibody binding. Since this antibody targets a specific region around Lysine 118, researchers should verify that post-translational modifications near this site (such as methylation, acetylation, or phosphorylation) won't affect antibody recognition. For co-detection studies with other histone markers, antibody compatibility in multiplexed assays should be tested, particularly regarding species of origin to avoid cross-reactivity in secondary detection systems.

How can this antibody be applied in cancer research contexts?

The HIST1H2AG (Ab-118) Antibody has demonstrated utility in cancer research, as evidenced by its successful application in immunohistochemistry of paraffin-embedded human breast cancer and glioma tissues . Researchers can use this antibody to investigate alterations in histone H2A expression or localization in tumor samples compared to normal tissues. This may provide insights into epigenetic dysregulation associated with tumorigenesis or cancer progression. The antibody can also be valuable in studying histone dynamics in cancer cell lines using techniques like immunofluorescence, as demonstrated in HeLa cells .

What methodological approaches can enhance ChIP efficiency with this antibody?

To optimize ChIP experiments using the HIST1H2AG (Ab-118) Antibody, researchers should:

• Perform careful crosslinking optimization (typically 1% formaldehyde for 10-15 minutes)

• Ensure thorough sonication to generate DNA fragments of 200-500 bp

• Include appropriate blocking agents to reduce non-specific binding

• Optimize antibody concentration through preliminary titration experiments

• Include proper controls (IgG negative control and positive control using a well-characterized histone antibody)

• Consider using specialized ChIP-grade protease inhibitors to preserve epitope integrity

These methodological refinements can significantly improve signal-to-noise ratio and reproducibility in ChIP experiments targeting histone H2A.

What are common causes of background signal when using this antibody in immunohistochemistry applications?

High background in IHC applications with the HIST1H2AG (Ab-118) Antibody may result from several factors:

• Insufficient blocking: Extend blocking time or use alternative blocking reagents (5% BSA, 5% normal serum)

• Excessive primary antibody concentration: Adhere to the recommended dilution range (1:20-200) and optimize through titration

• Inadequate washing: Increase wash duration or frequency between incubation steps

• Endogenous peroxidase/phosphatase activity: Ensure proper quenching steps prior to antibody incubation

• Non-specific binding to highly charged tissues: Consider adding 0.1-0.3M NaCl to antibody diluent

• Tissue fixation artifacts: Optimize fixation protocols or consider antigen retrieval methods

Each potential source should be systematically addressed to improve signal specificity in IHC applications.

How can specificity of the antibody be validated in experimental systems?

Researchers should validate the specificity of the HIST1H2AG (Ab-118) Antibody through multiple approaches:

• Western blot analysis showing a band of the expected molecular weight (around 14 kDa for histone H2A)

• Peptide competition assays where pre-incubation with the immunizing peptide blocks specific signal

• Genetic knockdown/knockout validation where signal reduction corresponds to decreased target expression

• Correlation with orthogonal detection methods (e.g., mass spectrometry)

• Comparison with other validated antibodies targeting different epitopes of the same protein

These validation strategies help ensure experimental results reflect genuine biological phenomena rather than antibody artifacts.

What are the most effective antigen retrieval methods for IHC applications with this antibody?

For optimal immunohistochemical detection using the HIST1H2AG (Ab-118) Antibody, heat-induced epitope retrieval (HIER) methods are recommended. The most effective approaches include:

• Citrate buffer (pH 6.0) heating: 10mM sodium citrate buffer heated to 95-100°C for 20 minutes

• EDTA buffer (pH 8.0) retrieval: 1mM EDTA heated to 95-100°C for 20 minutes

• Tris-EDTA (pH 9.0) method: 10mM Tris, 1mM EDTA heated to 95-100°C for 20 minutes

The optimal method should be determined empirically, as histone epitopes can respond differently to retrieval conditions depending on fixation parameters and tissue type. Generally, EDTA-based methods may provide better results for nuclear antigens like histones.

How should researchers address potential cross-reactivity with other histone H2A variants?

Due to the high sequence homology between histone H2A variants, cross-reactivity may occur with this antibody. Researchers can address this by:

• Performing parallel detection with variant-specific antibodies to identify potential overlapping signals

• Utilizing recombinant histone H2A variants in competition assays to assess relative binding affinities

• Implementing mass spectrometry validation to definitively identify detected protein species

• Conducting siRNA knockdown experiments targeting specific H2A variants to determine contribution to observed signals

• Comparing immunoblot patterns with predicted molecular weights of different H2A variants

These approaches help distinguish between specific detection of HIST1H2AG and potential cross-reactivity with related histone variants.

How should experiments be designed to study dynamic changes in H2A distribution during cell cycle progression?

To effectively study H2A dynamics throughout the cell cycle using the HIST1H2AG (Ab-118) Antibody, researchers should:

• Implement cell synchronization techniques (such as double thymidine block, nocodazole arrest, or serum starvation/stimulation)

• Collect samples at defined time points representing distinct cell cycle phases

• Combine immunofluorescence detection of HIST1H2AG with cell cycle markers (e.g., phospho-histone H3 for mitosis)

• Utilize flow cytometry with DNA content analysis to correlate H2A staining with cell cycle position

• Consider ChIP-seq approaches at different cell cycle stages to map genome-wide H2A distribution changes

• Include live-cell imaging with fluorescently tagged H2A to complement antibody-based fixed-cell analysis

This multi-parameter approach allows for comprehensive analysis of both spatial and temporal dynamics of histone H2A throughout cell division.

What controls should be included when using this antibody in ChIP experiments?

A robust ChIP experimental design using the HIST1H2AG (Ab-118) Antibody should include the following controls:

• Input control: Non-immunoprecipitated chromatin representing total DNA before IP (typically 5-10%)

• Negative control: IgG from the same species as the primary antibody (rabbit)

• Positive control: Known regions where H2A is expected (such as actively transcribed genes)

• Negative genomic regions: Areas known to have low H2A association

• Technical replicates: Multiple immunoprecipitations from the same chromatin preparation

• Biological replicates: ChIP performed on independently prepared samples

• Antibody titration controls: To determine optimal antibody concentration

These controls ensure experimental validity and facilitate accurate interpretation of ChIP results when studying histone H2A genomic distribution.

How can researchers effectively combine this antibody with other histone modification markers in multiplexed imaging?

For successful multiplexed imaging of HIST1H2AG alongside other histone modifications, researchers should:

• Select antibodies raised in different host species to enable species-specific secondary detection

• If using multiple rabbit antibodies, employ sequential immunostaining with complete stripping between rounds

• Utilize directly conjugated primary antibodies with non-overlapping fluorophores

• Implement spectral imaging and unmixing techniques to resolve closely positioned signals

• Include single-stained controls to establish bleed-through parameters

• Consider tyramide signal amplification for weak signals while maintaining multiplexing capability

• Validate antibody combinations for potential steric hindrance effects when epitopes are in close proximity

These approaches enable simultaneous visualization of histone H2A and its associated modifications, providing spatial context for epigenetic regulation studies.

How should researchers interpret variations in staining intensity between different cell types?

When interpreting differential HIST1H2AG staining patterns across cell types, researchers should consider:

• Intrinsic biological differences in histone H2A expression levels between cell lineages

• Cell-type specific chromatin compaction states affecting epitope accessibility

• Variations in histone post-translational modifications that might influence antibody binding

• Technical factors such as fixation efficiency differences between cell types

• Cell cycle distribution differences in heterogeneous populations

• Nuclear architecture variations that affect apparent signal intensity

Quantitative approaches like fluorescence intensity measurements normalized to nuclear area or DNA content can help standardize comparisons between cell types. Validation through orthogonal methods such as western blotting of sorted populations is recommended for confirming observed differences.

What methodological approaches can address contradictory results between ChIP-seq and immunofluorescence data?

When confronted with discrepancies between HIST1H2AG distribution patterns in ChIP-seq versus immunofluorescence experiments, researchers should:

• Evaluate fixation differences between methods (formaldehyde for ChIP vs. paraformaldehyde/methanol for IF)

• Consider epitope accessibility variances in different experimental contexts

• Assess ChIP resolution limitations compared to the spatial resolution of microscopy

• Implement ChIP-seq normalization strategies to account for technical biases

• Use alternative antibodies targeting different epitopes of the same protein

• Employ super-resolution microscopy to better align microscopy data with genomic resolution

• Consider complementary approaches like CUT&RUN or CUT&Tag that offer advantages of both techniques

These methodological considerations help reconcile seemingly contradictory results across platforms and provide a more complete understanding of histone H2A biology.

How can computational approaches enhance analysis of HIST1H2AG distribution patterns?

Advanced computational methods can significantly enhance the analysis of data generated using the HIST1H2AG (Ab-118) Antibody:

• For ChIP-seq data:

  • Peak calling algorithms optimized for broad histone signals

  • Differential binding analysis across experimental conditions

  • Integration with other epigenomic datasets (DNA methylation, chromatin accessibility)

  • Motif enrichment analysis to identify co-occurring regulatory elements

• For microscopy data:

  • Automated nuclear segmentation for high-throughput analysis

  • Intensity correlation analysis with other nuclear markers

  • Spatial statistics to quantify distribution patterns

  • Machine learning classification of nuclear phenotypes

These computational approaches enable extraction of biologically meaningful patterns from complex datasets, facilitating discovery of novel insights into histone H2A function and regulation.