Acetyl-Histone H2A type 1-B/E (K9) Recombinant Monoclonal Antibody

What is Histone H2A type 1-B/E and why is K9 acetylation significant?

Histone H2A type 1-B/E is a core component of nucleosomes, which wrap and compact DNA into chromatin. Nucleosomes limit DNA accessibility to cellular machineries requiring DNA as a template, thereby playing a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability . The acetylation of lysine 9 (K9) on H2A type 1-B/E is primarily associated with transcriptional activation. This modification helps recruit transcriptional activators, coactivators, and chromatin remodeling complexes to gene promoters, enhancing transcription initiation and progression . Acetylation at K9 is also involved in DNA repair mechanisms and epigenetic signaling pathways that coordinate gene regulation.

What are the primary validated applications for Acetyl-Histone H2A type 1-B/E (K9) antibodies?

Based on validation studies, this recombinant monoclonal antibody has been successfully employed in multiple applications:

Immunocytochemical and immunofluorescent staining of HeLa cells has been demonstrated with this antibody at dilutions of 1:100 and 1:56, respectively .

How does this recombinant monoclonal antibody differ from polyclonal alternatives?

The Acetyl-Histone H2A type 1-B/E (K9) recombinant monoclonal antibody (clone 3H5) offers several advantages over polyclonal antibodies:

• Consistency: Being a monoclonal antibody derived from a single clone (3H5), it provides superior batch-to-batch consistency compared to polyclonal alternatives .

• Specificity: This antibody recognizes a single epitope (acetylated K9 of H2A type 1-B/E), resulting in higher specificity than polyclonal antibodies that might recognize multiple epitopes .

• Production method: The recombinant production involves cloning genes encoding both heavy and light chains of the antibody, inserting them into expression vectors, and introducing them into host cells for antibody production and secretion . This controlled process ensures reproducibility.

• Purification: The antibody undergoes affinity chromatography purification, ensuring high purity and reduced non-specific binding .

What are the optimal sample preparation protocols for using this antibody in cellular studies?

For optimal results in cellular applications, follow these methodological guidelines:

For Immunocytochemistry (ICC):

• Fix cells using 4% paraformaldehyde for 15 minutes at room temperature.

• Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes.

• Block with 5% normal goat serum in PBS for 1 hour.

• Incubate with the primary antibody at 1:100 dilution overnight at 4°C.

• Wash three times with PBS (5 minutes each).

• Incubate with appropriate secondary antibody.

• Counterstain nuclei with DAPI if desired .

For Immunofluorescence (IF):

• Follow the same fixation and permeabilization steps as for ICC.

• Use the antibody at 1:56 dilution for optimal results.

• Use Alexa Fluor 488-conjugated goat anti-rabbit IgG as secondary antibody.

• DAPI counterstain has been successfully used to visualize nuclei .

How should this antibody be stored to maintain its effectiveness?

To ensure optimal antibody performance over time, adhere to these storage protocols:

• Storage temperature: Store at -20°C or -80°C .

• Aliquoting: Divide into single-use aliquots to avoid repeated freeze-thaw cycles, which can degrade antibody performance .

• Storage buffer: The antibody is provided in PBS (pH 7.4) containing 150mM NaCl, 50% glycerol, and 0.02% sodium azide .

• Handling precautions: Note that the product contains sodium azide, which is a poisonous and hazardous substance that should be handled by trained staff only .

If a slight precipitate forms during storage, it can be dissolved by gently vortexing the solution. This will not interfere with antibody performance .

How can I validate the specificity of this antibody in my experimental system?

To ensure the antibody is specifically detecting acetylated H2A K9 in your system:

• Peptide competition assay: Pre-incubate the antibody with the synthetic acetylated peptide used as immunogen (corresponding to residues surrounding K9 of human histone H2A type 1-B/E) . This should abolish specific staining.

• HDAC inhibitor treatment: Treat cells with HDAC inhibitors like trichostatin A (TSA) or sodium butyrate to increase global histone acetylation. This should increase signal intensity if the antibody is specific.

• Knockout/knockdown controls: Use cells where the target histone or the acetyltransferase responsible for K9 acetylation has been depleted.

• Western blot analysis: Perform western blotting with nuclear extracts to confirm the antibody detects a single band at approximately 14 kDa, which is the expected molecular weight of histone H2A .

What are common causes of high background when using this antibody?

High background can compromise data interpretation. Address these common causes methodologically:

• Insufficient blocking: Increase blocking time or use a different blocking agent (BSA, normal serum, or commercial blockers).

• Antibody concentration: The working dilution may be too concentrated. Test a dilution series:

  • For ICC: Try 1:200-1:500 dilutions

  • For IF: Try 1:100-1:200 dilutions

• Fixation issues: Overfixation can increase background. Optimize fixation time or try alternative fixatives.

• Secondary antibody cross-reactivity: Use secondary antibodies specifically validated for your application and species. Consider pre-adsorbed secondary antibodies to reduce cross-reactivity.

• Autofluorescence: For IF applications, include an unstained control to assess natural autofluorescence of your samples.

How can I optimize signal-to-noise ratio in chromatin studies using this antibody?

To enhance specific signal while minimizing background noise:

• Titrate antibody concentration: Determine the minimum antibody concentration that yields maximum specific signal. Start with manufacturer's recommended dilutions and adjust based on results .

• Sample preparation: For chromatin studies, ensure thorough crosslinking (if applicable) and appropriate chromatin fragmentation.

• Washing stringency: Increase the number of washes or add low concentrations of detergents (0.05-0.1% Tween-20) to washing buffers.

• Signal amplification systems: Consider tyramide signal amplification (TSA) for applications requiring enhanced sensitivity.

• Image acquisition parameters: When capturing fluorescence images, optimize exposure times and gain settings to maximize signal while avoiding saturation.

How does H2A K9 acetylation function within the broader context of histone modifications?

H2A K9 acetylation operates within a complex network of histone modifications:

• Integration with histone code: H2A K9 acetylation works in concert with other modifications like H3K27 acetylation to promote gene activation. None of the residues that are targets of post-translational modification in major-type H2A (acetylation, phosphorylation, and ubiquitination) are present in certain H2A variants like H2A.Bbd, suggesting specialized regulatory mechanisms .

• Nucleosome stability: Acetylation of histones, including H2A K9, can affect nucleosome stability. Studies show that H2A.B (a variant of H2A) forms the most dynamic nucleosomes of any H2A variant . This impacts chromatin accessibility and gene regulation.

• Variant-specific functions: Different H2A variants show distinct patterns of post-translational modifications. The H2A.Z variant is widely distributed throughout the genome, especially in promoter and enhancer regions, and is essential to nucleosome turnover, DNA repair, heterochromatin formation, and gene transcription .

How can this antibody be used in ChIP or ChIP-seq experiments to study genome-wide distribution of H2A K9 acetylation?

For Chromatin Immunoprecipitation (ChIP) applications:

• Protocol optimization:

  • Crosslinking: Use 1% formaldehyde for 10 minutes at room temperature

  • Sonication: Optimize to achieve 200-500 bp fragments

  • Antibody amount: Start with 2-5 μg per ChIP reaction

  • Incubation: Overnight at 4°C with rotation

• Quality controls:

  • Input samples: Include 5-10% input control

  • Positive controls: Target known regions with high H2A K9 acetylation

  • Negative controls: Use IgG control and target gene deserts

• Sequential ChIP: To study co-occurrence with other modifications, perform sequential ChIP with antibodies against other histone marks.

• Data analysis considerations:

  • For ChIP-seq, acetylation marks typically appear as broad peaks rather than sharp binding sites

  • Compare distribution with known enhancers, promoters, and transcriptionally active regions

  • Correlate with transcriptome data to establish functional relevance

What is known about the relationship between H2A variant incorporation and disease states?

The replacement of canonical histones by histone H2A variants is pivotal in gene regulation and DNA damage repair, with implications for various diseases:

• Cancer: Dysregulation of H2A variants and their modifications has been implicated in multiple cancers. For example, aberrant H2A.Z distribution correlates with altered gene expression in various malignancies .

• Neurodegenerative disorders: Studies in the mouse hippocampus show that H2A.B (a variant of H2A) is situated in the transcription start site region and is positively related to gene transcription activation , suggesting potential roles in neurological function and dysfunction.

• Developmental disorders: H2A.Z has been shown to be essential in organisms including mouse, fly, frog, and tetrahymena, indicating its crucial role in development . Disruption of H2A variant incorporation or modification can lead to developmental abnormalities.

• Research tools: Antibodies like the Acetyl-Histone H2A type 1-B/E (K9) Recombinant Monoclonal Antibody provide valuable tools for studying these disease connections, allowing researchers to map the distribution and dynamics of specific histone modifications in disease models and patient samples.

How can this antibody be integrated into multiplexed epigenetic profiling approaches?

Modern epigenetic research often requires simultaneous analysis of multiple modifications:

• Sequential immunofluorescence: This antibody can be used in sequential IF protocols where multiple rounds of antibody staining, imaging, and elution allow detection of numerous marks on the same sample.

• Mass cytometry applications: The conjugation-ready format makes this antibody ideal for use in mass cytometry (CyTOF) applications, where metal-conjugated antibodies can be used to simultaneously detect multiple histone modifications at the single-cell level .

• Multiplexed imaging: The antibody can be incorporated into multiplexed imaging techniques:

  • Cyclic immunofluorescence (CycIF)

  • CO-Detection by indEXing (CODEX)

  • Multiplexed ion beam imaging (MIBI)

• Matched antibody pairs: This antibody is available as part of matched antibody pairs (e.g., as capture and detection antibodies in Cytometric bead arrays) , facilitating multiplexed analysis of histone modifications in complex samples.