HIST1H4A (Ab-44) Antibody

What is HIST1H4A and what is its biological role?

HIST1H4A is a gene encoding Histone H4, which functions as a core component of nucleosomes. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to cellular machineries that use DNA as a template. Histones play central roles in transcription regulation, DNA repair, DNA replication, and chromosomal stability. DNA accessibility is regulated through a complex set of post-translational modifications of histones (the "histone code") and through nucleosome remodeling . Histone H4 is essential for chromatin structure and is highly conserved across species, making it a crucial target for understanding epigenetic regulation.

What applications has HIST1H4A (Ab-44) Antibody been validated for?

According to the product information, HIST1H4A (Ab-44) Antibody has been tested and validated for ELISA and immunohistochemistry (IHC) applications. The recommended dilution for IHC applications is 1:10-1:100 . This antibody is raised in rabbits and is a polyclonal antibody that specifically targets the peptide sequence around the Lysine-44 site of human Histone H4. It recognizes human samples and is provided in liquid form with 50% glycerol and 0.01M PBS (pH 7.4) buffer containing 0.03% Proclin 300 as a preservative .

How should HIST1H4A (Ab-44) Antibody be stored and handled?

The antibody should be stored at -20°C or -80°C upon receipt to maintain its efficacy. Repeated freeze-thaw cycles should be avoided as they can compromise antibody performance . The antibody is supplied in a liquid form with preservatives (0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4), which help maintain its activity during storage. For optimal results, aliquoting the antibody before freezing is recommended to minimize freeze-thaw cycles when using it for multiple experiments.

How does HIST1H4A (Ab-44) Antibody compare to other Histone H4 antibodies?

The HIST1H4A (Ab-44) Antibody targets specifically the region around Lysine-44 of human Histone H4, which distinguishes it from other Histone H4 antibodies that may target different epitopes. Other Histone H4 antibodies available include recombinant options like the Rabbit Anti-HIST1H4A Recombinant Antibody (clone CBACN-691) suitable for WB and IHC , and fluorescent conjugated options like the Histone H4 antibody (CL488-16047) validated for IF/ICC and flow cytometry . Selection between these antibodies should be based on specific research needs, target species, and intended applications.

What role does histone H4 play in cancer epigenetics and how can HIST1H4A antibodies contribute to this research?

Recent discoveries have highlighted the oncogenic role of histone mutations as one of the most relevant findings in cancer epigenetics . Histone H4 is a key component in chromatin regulation, and alterations in histone modifications or chromatin remodeling are implicated in tumor development and progression. Aberrant expression or incorporation of histone variants has been linked to more aggressive cancer phenotypes . HIST1H4A antibodies, including the Ab-44, can be valuable tools for investigating these epigenetic alterations by enabling researchers to:

• Detect changes in histone H4 expression levels in tumor samples

• Study specific histone modifications at the Lys-44 position and surrounding regions

• Investigate chromatin structure alterations in cancer cells

• Examine the association between histone H4 and oncogenic pathways

These applications make HIST1H4A antibodies essential for advancing our understanding of cancer epigenetics and potentially identifying new therapeutic targets.

How can the HIST1H4A (Ab-44) Antibody be incorporated into multi-parameter epigenetic studies?

For comprehensive epigenetic research, HIST1H4A (Ab-44) Antibody can be integrated into multi-parameter studies through several approaches:

• Sequential Immunostaining: When paired with antibodies against other histone modifications or chromatin-associated proteins, researchers can perform sequential staining on the same tissue section. This requires careful optimization of antibody dilutions (IHC: 1:10-1:100 for HIST1H4A Ab-44) and antigen retrieval methods.

• Complementary Techniques: The antibody can be used in conjunction with:

  • Chromatin immunoprecipitation (ChIP) to analyze histone H4 binding sites

  • Mass spectrometry to identify and quantify histone modifications

  • RNA-seq to correlate chromatin states with gene expression

• Immunofluorescence Co-localization: When studying spatial relationships between histone H4 and other nuclear proteins, this antibody can be paired with fluorescent-conjugated secondary antibodies or compared with ready-made fluorescent histone H4 antibodies like the CL488-16047, which has excitation/emission maxima wavelengths of 493 nm/522 nm .

What are the considerations for using HIST1H4A (Ab-44) Antibody in studying post-translational modifications of histones?

When utilizing HIST1H4A (Ab-44) Antibody to study post-translational modifications (PTMs), researchers should consider:

• Epitope Accessibility: Since the antibody targets the region around Lysine-44, modifications at or near this site might affect antibody binding. Acetylation, methylation, or other PTMs at Lys-44 could potentially mask the epitope.

• Fixation Methods: Different fixation protocols can preserve or expose certain histone modifications differently. For optimal detection of specific PTMs, fixation methods should be carefully selected and standardized.

• Comparison Controls: Include appropriate controls, such as:

  • Samples treated with histone deacetylase inhibitors

  • Samples with known modification status

  • Recombinant histones with defined modification patterns

• Complementary Modification-Specific Antibodies: To comprehensively study the "histone code," pair the HIST1H4A (Ab-44) Antibody with modification-specific antibodies targeting known PTM sites on histone H4, such as acetylation at K5, K8, K12, and K16, or methylation at various arginine residues.

What are the optimal conditions for immunohistochemistry using HIST1H4A (Ab-44) Antibody?

For optimal IHC results with HIST1H4A (Ab-44) Antibody, consider the following protocol recommendations:

• Dilution Range: The manufacturer recommends a dilution range of 1:10-1:100 for IHC applications . Testing multiple dilutions is advisable for optimizing signal-to-noise ratio in your specific tissue type.

• Antigen Retrieval: Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 8.0) is typically effective for histone antibodies. Compare multiple methods to determine optimal conditions for your specific samples.

• Blocking: Use a blocking solution containing 5-10% normal serum (from the same species as the secondary antibody) and 1% BSA to reduce background staining.

• Incubation:

  • Primary antibody (HIST1H4A Ab-44): Incubate overnight at 4°C or 1-2 hours at room temperature

  • Secondary antibody: Typically 30-60 minutes at room temperature

• Detection System: For polyclonal rabbit antibodies like HIST1H4A (Ab-44), a polymer-based detection system often provides better sensitivity than biotin-avidin systems.

• Counterstaining: Light hematoxylin counterstain works well for nuclear antigens like histone H4.

How can specificity of the HIST1H4A (Ab-44) Antibody be validated in experimental settings?

Validating antibody specificity is crucial for reliable research. For HIST1H4A (Ab-44) Antibody, consider these validation approaches:

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide (sequence around Lys-44 of human Histone H4) before application to samples. Signal elimination confirms specificity.

• Knockout/Knockdown Controls: Though complete H4 knockout is lethal, RNAi-mediated knockdown of specific H4 variants can serve as partial controls.

• Recombinant Protein Controls: Test the antibody against recombinant H4 proteins and variants to confirm target recognition.

• Cross-Reactivity Assessment: Test the antibody on samples from multiple species. While HIST1H4A (Ab-44) is specified for human reactivity , histone H4 is highly conserved, so cross-reactivity with other species might occur.

• Parallel Antibody Comparison: Compare staining patterns with other validated H4 antibodies, such as the recombinant antibodies mentioned in the search results .

• Western Blot Validation: Though not specifically listed in the tested applications, Western blot can verify that the antibody recognizes protein of the expected molecular weight (observed molecular weight: 14 kDa and 11 kDa) .

What are common troubleshooting strategies when using HIST1H4A (Ab-44) Antibody in immunofluorescence applications?

When adapting the HIST1H4A (Ab-44) Antibody for immunofluorescence applications, researchers might encounter several challenges. Here are troubleshooting strategies for common issues:

• Weak or No Signal:

  • Increase antibody concentration (try higher end of recommended range: closer to 1:10)

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

  • Enhance antigen retrieval (increase time or try alternative buffers)

  • Ensure proper storage of antibody (avoid repeated freeze-thaw cycles)

• High Background:

  • Increase blocking time and concentration (5-10% normal serum with 1% BSA)

  • Reduce primary antibody concentration (move toward 1:100 dilution)

  • Add 0.1-0.3% Triton X-100 for better penetration and reduced non-specific binding

  • Include additional washing steps with 0.1% Tween-20 in PBS

• Non-Nuclear Staining (unexpected for histone proteins):

  • Verify fixation protocol (4% paraformaldehyde is typically effective)

  • Ensure permeabilization is adequate for nuclear antigen access

  • Control for autofluorescence using unstained controls

• Inconsistent Results Between Experiments:

  • Standardize fixation time and conditions

  • Prepare single-use aliquots to avoid freeze-thaw cycles

  • Document lot numbers as antibody performance can vary between lots

• Cross-Reactivity Issues:

  • Increase washing stringency with higher salt concentration

  • Pre-absorb antibody with non-specific proteins

  • Consider secondary antibody alternatives

How should quantitative data from HIST1H4A (Ab-44) Antibody experiments be normalized and analyzed?

For rigorous quantitative analysis of data obtained using HIST1H4A (Ab-44) Antibody, consider these methodological approaches:

• Immunohistochemistry/Immunofluorescence Quantification:

  • Use digital image analysis software (ImageJ, QuPath, etc.) to quantify signal intensity

  • Normalize nuclear H4 staining to total nuclear area (DAPI or hematoxylin counterstain)

  • Apply H-score or Allred scoring systems for semi-quantitative analysis

  • Include calibration controls in each experiment for inter-experimental normalization

• Statistical Considerations:

  • Apply appropriate statistical tests based on data distribution (parametric vs. non-parametric)

  • For IHC studies, consider inter-observer variability by having multiple researchers score samples

  • Calculate intra-assay precision (CV% typically <8% for similar assays) and inter-assay precision (CV% typically <10%)

• Experimental Design for Quantification:

  • Include biological replicates (n≥3) and technical replicates

  • Design experiments with appropriate positive and negative controls

  • Consider performing dilution linearity tests similar to those reported for HIST1H4A ELISA (1:1, 1:2, 1:4, 1:8 dilutions)

• Data Presentation:

  • Present normalized data with appropriate error bars

  • Include representative images alongside quantitative graphs

  • Report exact p-values and statistical methods

How do results from HIST1H4A (Ab-44) Antibody compare with data from ELISA-based histone H4 detection methods?

When comparing immunohistochemistry results using HIST1H4A (Ab-44) Antibody with ELISA-based methods for histone H4 detection, researchers should consider:

What are the implications of detecting different molecular weight forms of Histone H4 in experimental data?

Histone H4 can appear at different molecular weights in experimental data, which has important implications for data interpretation:

• Expected Molecular Weight Profile:

  • The calculated molecular weight of Histone H4 is approximately 11 kDa (102 amino acids)

  • Observed molecular weights can include both 11 kDa and 14 kDa bands

• Causes of Different Molecular Weight Forms:

  • Post-translational modifications (acetylation, methylation, phosphorylation) can alter apparent molecular weight

  • Histone variants with slightly different sequences

  • Proteolytic processing

  • Differential migration based on sample preparation conditions

• Analytical Implications:

  • Multiple bands may represent different modification states of Histone H4

  • The 14 kDa band observed with some antibodies may include H4 with extensive modifications

  • When comparing results across studies, consider whether total H4 or specific forms were detected

• Verification Strategies:

  • Use recombinant H4 with known modification status as controls

  • Employ modification-specific antibodies in parallel experiments

  • Consider mass spectrometry to characterize the specific forms detected

• Biological Significance:

  • Changes in the ratio between different molecular weight forms may indicate alterations in modification patterns

  • Cancer and other diseases often show altered histone modification profiles

  • Interpreting these changes can provide insights into disease mechanisms and potential therapeutic targets