HIST1H4A (Ab-79) Antibody

What is HIST1H4A and what role does it play in chromatin structure?

HIST1H4A belongs to the histone H4 family, which are basic nuclear proteins responsible for the nucleosome structure of chromosomal fiber in eukaryotes. Histones are fundamental components of chromatin, where two molecules of each core histone (H2A, H2B, H3, and H4) form an octamer around which approximately 146 bp of DNA wraps to create nucleosomes. HIST1H4A is intronless and is located within the large histone gene cluster on chromosome 6 . Unlike typical mRNA, transcripts from this gene lack polyA tails and instead contain a palindromic termination element . The protein plays a critical role in DNA packaging and chromosome condensation during cell division and influences gene expression through various post-translational modifications.

What applications is the HIST1H4A (Ab-79) antibody validated for?

The HIST1H4A (Ab-79) Polyclonal Antibody has been validated for multiple research applications that allow for diverse experimental approaches:

When designing experiments, researchers should note that this antibody is specifically developed for human samples and cross-reactivity with other species should be verified before use in comparative studies .

What is the specificity and target site of the HIST1H4A (Ab-79) antibody?

The HIST1H4A (Ab-79) antibody is designed to recognize and bind to a peptide sequence around the site of Lysine 79 (Lys79) derived from Human Histone H4 . This specificity is important for researchers studying histone modifications in this region. The antibody has been developed using rabbits as the host organism and is of IgG isotype . The polyclonal nature of this antibody means it can recognize multiple epitopes of the target antigen, potentially increasing sensitivity but requiring careful validation of specificity.

For researchers interested in post-translational modifications, it's worth noting that other specialized antibodies such as those detecting formylation at K91 are also available for histone H4 research , suggesting that researchers should carefully select the appropriate antibody based on the specific modification site they are investigating.

How should samples be prepared for optimal detection using HIST1H4A (Ab-79) antibody?

For optimal results with the HIST1H4A (Ab-79) antibody, sample preparation protocols should be tailored to the specific application:

For Western Blotting:

• Prepare cell lysates in a buffer containing protease inhibitors to prevent histone degradation

• For histone-specific preparation, consider acid extraction methods that enrich for basic proteins

• Load 15-25 μg of total protein per lane (based on NIH/3T3 cell lysate loading)

• Use SDS-PAGE with appropriate percentage gels for low molecular weight proteins (the predicted band size for histone H4 is approximately 11 kDa)

For ELISA Applications:

• Sample dilution should be carefully considered based on expected protein concentration

• Linearity of dilution studies indicate that serum samples show good recovery (95-108% at 1:1 dilution)

• Detection range for commercial ELISA kits is typically 37.5-2400 pg/mL with a sensitivity of 9.3 pg/mL

For Chromatin Immunoprecipitation:

• Crosslink proteins to DNA using formaldehyde (typically 1% for 10 minutes)

• Sonicate chromatin to appropriate fragment sizes (200-500 bp)

• Use 2-5 μg of antibody per immunoprecipitation reaction

• Include appropriate controls (input, IgG control, positive control target)

What controls should be included when working with HIST1H4A (Ab-79) antibody?

When designing experiments using the HIST1H4A (Ab-79) antibody, incorporating appropriate controls is essential for result interpretation and troubleshooting:

Positive Controls:

• Known histone H4-expressing cell lines (most eukaryotic cells express histones)

• Recombinant histone H4 protein

• Previously validated sample with confirmed histone H4 expression

Negative Controls:

• Secondary antibody only (no primary antibody) to assess background

• Isotype control (rabbit IgG) to identify non-specific binding

• Peptide competition assay using the immunizing peptide to confirm specificity

Application-Specific Controls:

• For ChIP: Input chromatin (pre-immunoprecipitation sample)

• For ELISA: Standard curve using recombinant HIST1H4A protein

• For IHC/IF: Tissue known to be negative for the target or with primary antibody omitted

Validation Controls:

• Histone H4 peptide array to confirm binding specificity to the target site (Lys79)

• Western blot showing predicted molecular weight (11 kDa)

What protein interactions have been identified for HIST1H4A and how might they influence experimental design?

HIST1H4A has been shown to interact with multiple proteins that may impact chromatin structure and function. Researchers should consider these interactions when designing experiments and interpreting results:

These interactions suggest that HIST1H4A may be involved in diverse cellular processes beyond its structural role in chromatin. The strong interaction with RAB5C (standardized value: 0.682774) indicates potential involvement in membrane trafficking pathways, while interaction with ATP5B suggests possible links to cellular energetics .

When designing co-immunoprecipitation experiments or studying chromatin complexes, researchers should consider these interaction partners as potential confounding factors or additional targets for investigation.

What are the methodological considerations for using HIST1H4A (Ab-79) antibody in ChIP assays?

Chromatin Immunoprecipitation (ChIP) using HIST1H4A (Ab-79) antibody requires specific methodological considerations to optimize results:

Chromatin Preparation:

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

• Sonication: Optimize sonication conditions to achieve DNA fragments of 200-500 bp

• Chromatin quality: Verify fragmentation by agarose gel electrophoresis before proceeding

Immunoprecipitation Protocol:

• Pre-clearing: Use protein A/G beads to reduce non-specific binding

• Antibody amount: Use 2-5 μg of HIST1H4A (Ab-79) antibody per reaction

• Incubation time: Overnight incubation at 4°C generally yields optimal results

• Washing stringency: Use increasingly stringent wash buffers to reduce background

Data Analysis Considerations:

• Normalization: Compare enrichment to input chromatin and IgG control

• Positive control loci: Include primers for known histone H4-enriched genomic regions

• Sequencing considerations: For ChIP-seq, ensure sufficient sequencing depth (>20 million reads)

How can researchers effectively study post-translational modifications of HIST1H4A?

Histone H4 undergoes numerous post-translational modifications (PTMs) that regulate chromatin structure and function. To effectively study these modifications:

Selection of Modification-Specific Antibodies:

• For lysine 79 modifications: Use the HIST1H4A (Ab-79) antibody as it targets this region

• For other modifications: Select antibodies specific to the modification of interest (e.g., formylation at K91)

• Validate antibody specificity against peptide arrays containing modified and unmodified peptides

Experimental Approaches:

• Western blotting: Use modification-specific antibodies alongside total H4 antibodies

• Mass spectrometry: For unbiased identification of multiple PTMs simultaneously

• ChIP-seq: Map genome-wide distribution of specific histone modifications

• Sequential ChIP (Re-ChIP): Identify genomic regions containing multiple modifications

Quantification Methods:

• ELISA assays: Provide quantitative measurement of specific modifications with sensitivity down to 9.3 pg/mL

• Protein arrays: Allow multiplexed detection of multiple histone modifications

• Imaging approaches: Visualize spatial distribution of modifications within nuclei

When studying histone modifications, it's crucial to consider the dynamic nature of these marks and their potential crosstalk. For example, modifications at one residue may influence the accessibility or recognition of nearby sites by modifying enzymes or antibodies.

What challenges might researchers encounter when using HIST1H4A (Ab-79) antibody in different experimental systems?

When working with HIST1H4A (Ab-79) antibody across different experimental systems, researchers should be aware of several potential challenges:

Cross-Reactivity Considerations:

• Species specificity: The antibody is validated for human samples but may show variable cross-reactivity with other species

• Histone variant recognition: Given the high sequence conservation among H4 variants, verify specificity against closely related proteins

• Epitope masking: Post-translational modifications near Lys79 may affect antibody binding

Technical Challenges by Application:

• Western Blotting:

  • Low molecular weight of histones (11 kDa) requires appropriate gel percentage

  • Histones are highly basic proteins that may need specialized transfer conditions

• Immunohistochemistry:

  • Fixation methods significantly impact histone epitope accessibility

  • Nuclear localization requires careful permeabilization optimization

• ELISA:

  • Matrix effects from different sample types may affect detection

  • Linear detection range in serum samples is 37.5-2400 pg/mL

Reproducibility Considerations:

• Lot-to-lot variation: Polyclonal antibodies may show batch variation

• Sample preparation consistency: Histone extraction methods should be standardized

• Quantification challenges: ELISA kits show intra-assay precision (CV<8%) and inter-assay precision (CV<10%)

How can researchers integrate HIST1H4A studies with broader epigenetic research?

Integrating HIST1H4A research into broader epigenetic studies requires consideration of multiple molecular mechanisms and analytical approaches:

Multi-Omics Integration Strategies:

• Combine ChIP-seq data using HIST1H4A (Ab-79) antibody with:

  • RNA-seq to correlate histone modifications with gene expression

  • ATAC-seq to assess chromatin accessibility

  • DNA methylation profiling to understand epigenetic crosstalk

Functional Analysis Approaches:

• CRISPR-based histone modification: Target specific modifications at HIST1H4A

• Histone demethylase/methyltransferase inhibitors: Assess impact on HIST1H4A modifications

• Single-cell approaches: Investigate cell-to-cell variability in HIST1H4A modifications

Biological Context Considerations:

• Cell cycle analysis: HIST1H4A modifications change during different cell cycle phases

• Development and differentiation: Study dynamic changes during cellular differentiation

• Disease models: Compare HIST1H4A modifications in normal vs. pathological states

Data Analysis Frameworks:

• Enrichment analysis: Identify biological pathways associated with HIST1H4A-bound regions

• Motif analysis: Determine transcription factors that co-localize with HIST1H4A modifications

• Chromosome conformation data integration: Connect HIST1H4A modifications with 3D genome organization

When examining protein interactions, researchers should note that HIST1H4A interacts with diverse proteins including chromatin components (HIST1H2BL), metabolic enzymes (ATP5B, ATP5A1), and signaling molecules (RAB5C) , suggesting involvement in multiple cellular processes beyond chromatin structure.

What are common troubleshooting strategies for experiments using HIST1H4A (Ab-79) antibody?

When experiments with HIST1H4A (Ab-79) antibody don't yield expected results, consider these troubleshooting approaches:

For Weak or No Signal:

• Antibody concentration: Titrate antibody to determine optimal concentration

• Epitope accessibility: Modify fixation/permeabilization protocols to improve antigen retrieval

• Sample preparation: Ensure histones are properly extracted and not degraded

• Detection system: Verify secondary antibody functionality and consider signal amplification

For High Background:

• Blocking optimization: Test different blocking reagents (BSA, milk, serum)

• Washing stringency: Increase number and duration of wash steps

• Antibody specificity: Perform peptide competition assay to verify specific binding

• Sample quality: Reduce endogenous peroxidase activity or autofluorescence

For Unexpected Results:

• Positive controls: Include known HIST1H4A-expressing samples

• Band size verification: Confirm correct molecular weight (11 kDa for histone H4)

• Lot testing: Compare results across different antibody lots

• Protocol consistency: Standardize all steps of the experimental workflow

How should researchers validate the specificity and performance of HIST1H4A (Ab-79) antibody?

Comprehensive validation of HIST1H4A (Ab-79) antibody ensures reliable experimental outcomes:

Specificity Validation:

• Peptide array testing: Verify binding to target peptide versus related sequences

• Western blot: Confirm single band at expected molecular weight (11 kDa)

• Knockdown/knockout controls: Reduced signal in HIST1H4A-depleted samples

• Peptide competition: Pre-incubation with immunizing peptide should abolish signal

Performance Validation:

• Sensitivity assessment: Determine lower limit of detection (ELISA sensitivity: 9.3 pg/mL)

• Dynamic range: Verify linear detection range (ELISA range: 37.5-2400 pg/mL)

• Reproducibility testing:

  • Intra-assay precision: CV<8% for repeated measurements within an assay

  • Inter-assay precision: CV<10% for measurements across different assays

Application-Specific Validation:

• For IHC/IF: Staining pattern should match known nuclear localization

• For ChIP: Enrichment at known histone H4-associated genomic regions

• For ELISA: Recovery testing across sample types (serum recovery: 93%, range 89-97%)

How is HIST1H4A research contributing to understanding chromatin dynamics?

Research utilizing tools like the HIST1H4A (Ab-79) antibody is advancing our understanding of chromatin dynamics in several key areas:

Nucleosome Assembly and Stability:

• HIST1H4A interactions with other histones, particularly HIST1H2BL (interaction value: 0.269638) , provide insights into nucleosome assembly mechanisms

• Studies of HIST1H4A modifications help explain how chromatin structure changes during cell cycle progression

Regulatory Networks:

• Interactions between HIST1H4A and non-histone proteins such as RAB5C (interaction value: 0.682774) suggest unexpected connections between chromatin and membrane trafficking pathways

• HIST1H4A association with metabolic enzymes like ATP5B (interaction value: 0.578784) points to potential links between cellular energetics and chromatin structure

Technological Advances:

• Antibodies targeting specific HIST1H4A modifications enable genome-wide mapping of epigenetic landscapes

• Increasing specificity of tools like the HIST1H4A (Ab-79) antibody allows for more precise dissection of histone variant functions

What emerging applications might benefit from HIST1H4A (Ab-79) antibody research?

Several cutting-edge research areas could be advanced through applications of HIST1H4A (Ab-79) antibody:

Single-Cell Epigenomics:

• Adaptation of ChIP protocols for single-cell analysis to study cell-to-cell variation in HIST1H4A modifications

• Integration with single-cell transcriptomics to correlate histone modifications with gene expression heterogeneity

Liquid Biopsy Development:

• Detection of circulating HIST1H4A in serum samples as potential biomarkers (ELISA sensitivity: 9.3 pg/mL)

• Monitoring dynamic changes in histone modifications during disease progression

Therapeutic Target Validation:

• Studying how experimental drugs affect HIST1H4A modifications and interactions

• Identifying specific HIST1H4A modifications associated with disease states

Developmental Biology:

• Mapping changes in HIST1H4A modifications during cellular differentiation

• Understanding the role of specific histone variants in developmental programming