HIST1H4A (Ab-91) Antibody

What is HIST1H4A and what is the target specificity of HIST1H4A (Ab-91) Antibody?

HIST1H4A is a histone H4 variant encoded by a gene located within histone cluster 1 on chromosome 6p22.1-p22.2 . The HIST1H4A (Ab-91) antibody is a rabbit polyclonal antibody specifically raised against the peptide sequence surrounding lysine 91 (K91) of human histone H4 . This antibody is designed to recognize the K91 region regardless of its modification state, making it valuable for general histone H4 detection. The antibody demonstrates reactivity with both human (Homo sapiens) and mouse (Mus musculus) samples, which reflects the high conservation of histone H4 sequences across species .

What is the significance of the lysine 91 residue in histone H4?

Lysine 91 in histone H4 (H4K91) represents a critical residue that undergoes several post-translational modifications with significant functional consequences. Research has shown that H4K91 can be monoubiquitylated by the E3 ligase BBAP . This modification appears to play a crucial role in the DNA damage response pathway, as BBAP-mediated monoubiquitylation of H4K91 is associated with subsequent mono- and dimethylation of H4K20, which are required for the recruitment of 53BP1 to DNA damage sites . Additionally, H4K91 can be acetylated (H4K91ac), and the balance between ubiquitylation and acetylation at this site appears to be regulated during DNA damage responses . The strategic position of K91 within the histone core makes it particularly important for nucleosome stability and chromatin structure.

What validated applications can HIST1H4A (Ab-91) Antibody be used for?

The HIST1H4A (Ab-91) Antibody has been validated for multiple experimental applications:

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of histone H4 in solution

• Western Blotting (WB): For detection of histone H4 in protein extracts

• Immunohistochemistry (IHC): For visualization of histone H4 in tissue sections

• Chromatin Immunoprecipitation (ChIP): For isolation of chromatin fragments containing histone H4

This versatility makes the antibody suitable for diverse experimental approaches in histone research, allowing for both qualitative and quantitative analyses of histone H4 in various sample types and experimental conditions.

How can HIST1H4A (Ab-91) Antibody be used to study histone H4 modifications in DNA damage response pathways?

The HIST1H4A (Ab-91) Antibody can serve as a valuable tool in DNA damage response studies, particularly when used in conjunction with modification-specific antibodies. Research has demonstrated that H4K91 monoubiquitylation by BBAP occurs in response to DNA damage induced by agents such as doxorubicin (Dox) . When investigating this pathway:

• Researchers should design time-course experiments following DNA damage induction to track changes in H4K91 modifications. The HIST1H4A (Ab-91) Antibody can establish baseline histone H4 levels, while modification-specific antibodies can track changes in H4K91 ubiquitylation and H4K20 methylation .

• Comparative ChIP experiments using HIST1H4A (Ab-91) Antibody alongside antibodies specific for ubiquitylated H4K91 and methylated H4K20 can reveal the temporal and spatial relationships between these modifications at DNA damage sites.

• Knockdown experiments targeting BBAP combined with immunoblotting using HIST1H4A (Ab-91) Antibody can help establish the dependency of specific H4 modifications on BBAP activity. Previous research has shown that BBAP depletion significantly decreases monoubiquitylated H4 and mono/dimethylated H4K20 while increasing H4K91 acetylation .

This integrated approach can provide comprehensive insights into how histone H4 modifications coordinate the DNA damage response.

What is the relationship between HIST1H4A expression and cell cycle progression?

Histone H4 expression is tightly regulated during the cell cycle, with significant implications for researchers studying cell proliferation and differentiation:

• During the S phase of the cell cycle, histone H4 expression is dramatically upregulated to accommodate the synthesis of new nucleosomes for the replicating genome. The HIST1H4A gene, being replication-dependent, shows peak expression during this phase .

• Research in 3T3-L1 preadipocytes has shown that knockdown of histone H4 using specific siRNA significantly impairs cell cycle progression, with more cells remaining in G0/G1 phase and fewer transitioning to S phase . This indicates that histone H4 expression is required for mitotic clonal expansion (MCE) during adipocyte differentiation.

• Transcriptional activation of histone H4 during MCE is mediated by the transcription factor C/EBPβ, which binds to specific sites in the histone H4 promoter region . ChIP experiments using both HIST1H4A (Ab-91) Antibody and anti-C/EBPβ antibodies can help elucidate this regulatory mechanism.

When studying the relationship between HIST1H4A expression and cell cycle progression, researchers should synchronize cells using established methods (serum starvation, thymidine block, etc.) and collect samples at defined time points for analysis with HIST1H4A (Ab-91) Antibody.

How does HIST1H4A (Ab-91) Antibody compare with modification-specific histone H4 antibodies in research applications?

The HIST1H4A (Ab-91) Antibody recognizes the sequence surrounding lysine 91 of histone H4 regardless of its modification state, whereas modification-specific antibodies detect specific post-translational modifications. This distinction is critical for experimental design:

For comprehensive studies of histone H4 biology, researchers should employ both types of antibodies: HIST1H4A (Ab-91) Antibody provides information on total H4 levels, while modification-specific antibodies reveal the dynamic changes in specific post-translational modifications . This approach is particularly valuable in time-course experiments following treatments that alter chromatin structure or in comparing different cell types or disease states.

What are the optimal protocols for using HIST1H4A (Ab-91) Antibody in Western blotting experiments?

For successful Western blotting with HIST1H4A (Ab-91) Antibody, researchers should follow these methodological guidelines:

• Sample Preparation:

  • Extract histones using acid extraction (0.2N HCl) or specialized histone extraction kits

  • For total cell lysates, use SDS lysis buffer with protease inhibitors and phosphatase inhibitors

  • Include 10-20 mM sodium butyrate to preserve acetylation modifications

  • Quantify protein concentration using Bradford or BCA assays

• Gel Electrophoresis:

  • Use 15-18% SDS-PAGE gels to achieve good separation of histones (~11-15 kDa)

  • Load 10-20 μg of acid-extracted histones or 30-50 μg of total cell lysate

  • Include molecular weight markers that cover the low molecular weight range

• Transfer and Blocking:

  • Transfer proteins to PVDF membrane (preferred over nitrocellulose for histones)

  • Use semi-dry transfer systems with methanol-containing transfer buffer

  • Block membranes with 5% non-fat dry milk or 3-5% BSA in TBST

• Antibody Incubation:

  • Dilute HIST1H4A (Ab-91) Antibody as recommended (typically 1:500 to 1:2000)

  • Incubate overnight at 4°C with gentle rocking

  • Wash extensively with TBST (at least 3 washes, 5-10 minutes each)

  • Use appropriate HRP-conjugated secondary antibody (anti-rabbit IgG)

• Detection and Controls:

  • Develop using enhanced chemiluminescence (ECL) reagents

  • Include positive controls (known histone H4-containing samples)

  • Include loading controls (total protein staining or house-keeping proteins)

  • For studying specific modifications, use modification-specific antibodies on parallel blots

This protocol should yield a clear band at approximately 11 kDa corresponding to histone H4.

What considerations are important for Chromatin Immunoprecipitation (ChIP) using HIST1H4A (Ab-91) Antibody?

Chromatin Immunoprecipitation with HIST1H4A (Ab-91) Antibody requires careful experimental design and optimization:

• Sample Preparation:

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

  • Quench with 125 mM glycine

  • Lyse cells and sonicate chromatin to generate fragments of 200-500 bp

  • Verify fragment size by agarose gel electrophoresis

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Use 2-5 μg of HIST1H4A (Ab-91) Antibody per ChIP reaction

  • Include appropriate controls:

    • IgG negative control

    • Input chromatin (non-immunoprecipitated)

    • Positive control (antibody against abundant histone mark)

  • Incubate overnight at 4°C with rotation

• Washing and Elution:

  • Wash immunoprecipitated complexes with increasingly stringent buffers

  • Elute chromatin from beads

  • Reverse crosslinks and purify DNA

• Analysis:

  • Perform qPCR targeting regions of interest

  • For genome-wide studies, prepare libraries for ChIP-seq

  • When investigating histone modifications at H4K91, perform sequential ChIP (re-ChIP) with modification-specific antibodies

• Data Interpretation:

  • The HIST1H4A (Ab-91) Antibody will pull down regions containing histone H4, which is expected to be broadly distributed across the genome

  • Enrichment patterns should be compared with those of modification-specific antibodies to determine the distribution of modified histones

For studying the relationship between transcription factors and histone H4, as demonstrated in research on C/EBPβ binding to histone H4 promoters, HIST1H4A (Ab-91) Antibody can be used alongside antibodies against relevant transcription factors in parallel ChIP experiments .

How can researchers validate the specificity of HIST1H4A (Ab-91) Antibody in their experimental systems?

Validating antibody specificity is crucial for ensuring reliable experimental results. For HIST1H4A (Ab-91) Antibody, researchers should implement multiple validation strategies:

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess immunizing peptide (sequence around K91 of histone H4)

  • Perform Western blotting or immunostaining with both blocked and unblocked antibody

  • Signal should be significantly reduced or eliminated with blocked antibody

• Knockdown/Knockout Validation:

  • Use siRNA or CRISPR-Cas9 to reduce or eliminate histone H4 expression

  • Compare antibody signal between control and knockdown/knockout samples

  • A specific antibody will show reduced signal in knockdown/knockout samples

  • Note: Complete histone H4 knockout may be lethal due to its essential role

• Multiple Antibody Comparison:

  • Test multiple antibodies targeting different epitopes of histone H4

  • Compare staining/binding patterns across different applications

  • Consistent patterns across antibodies suggest specific recognition

• Mass Spectrometry Validation:

  • Perform immunoprecipitation with HIST1H4A (Ab-91) Antibody

  • Analyze precipitated proteins by mass spectrometry

  • Confirm the presence of histone H4 and identify co-precipitating proteins

• Cross-Reactivity Testing:

  • Test the antibody against recombinant histone variants

  • Test reactivity with samples from different species

  • Confirm expected reactivity with human and mouse samples as specified

• Modification Status Verification:

  • Compare recognition patterns with modification-specific antibodies (H4K91ac, H4K91ub)

  • Verify that treatments known to affect K91 modifications (e.g., BBAP depletion, DNA damage) produce expected changes in signals from modification-specific antibodies while total H4 levels detected by HIST1H4A (Ab-91) Antibody remain stable

What are common issues when using HIST1H4A (Ab-91) Antibody in immunofluorescence experiments?

When performing immunofluorescence with HIST1H4A (Ab-91) Antibody, researchers might encounter several challenges:

• High Background Signal:

  • Cause: Insufficient blocking, excessive antibody concentration, or inadequate washing

  • Solution: Increase blocking time (2-3 hours), optimize antibody dilution (typically 1:50-1:200 for immunofluorescence) , and extend washing steps

• Weak or No Signal:

  • Cause: Epitope masking due to fixation, insufficient permeabilization, or antibody degradation

  • Solution: Try different fixation methods (paraformaldehyde vs. methanol), increase permeabilization time with 0.1-0.5% Triton X-100, and ensure proper antibody storage (aliquot and store at -20°C)

• Non-Nuclear Staining:

  • Cause: Non-specific antibody binding or cytoplasmic histone H4 (free or in autophagosomes)

  • Solution: Increase blocking stringency, pre-adsorb antibody, and include controls to distinguish between specific and non-specific staining

• Heterogeneous Staining Patterns:

  • Cause: Cell cycle-dependent expression of histone H4 or variable accessibility of the epitope

  • Solution: Synchronize cells or co-stain with cell cycle markers to correlate staining patterns with cell cycle phases

• Protocol Optimization:

  • Fixation: 4% paraformaldehyde for 15-20 minutes at room temperature

  • Permeabilization: 0.2% Triton X-100 for 10 minutes

  • Blocking: 5% BSA or 10% normal serum from the species of the secondary antibody

  • Antibody dilution: Start with 1:100 and optimize as needed

  • Incubation: Overnight at 4°C for primary antibody, 1-2 hours at room temperature for secondary

When studying specific modifications at H4K91, consider performing sequential or dual immunofluorescence with modification-specific antibodies to correlate total H4 with specific modifications.

How can HIST1H4A (Ab-91) Antibody be used to study the interplay between histone modifications in epigenetic regulation?

HIST1H4A (Ab-91) Antibody provides a valuable tool for investigating the complex relationships between different histone modifications:

• Sequential ChIP (Re-ChIP) Approaches:

  • First immunoprecipitate with HIST1H4A (Ab-91) Antibody

  • Elute the precipitated material

  • Perform a second immunoprecipitation with antibodies against specific modifications (H4K20me, H4K91ac)

  • This approach can reveal co-occurrence of modifications on the same histone molecules

• Multi-Omics Integration:

  • Combine ChIP-seq using HIST1H4A (Ab-91) Antibody with:

    • RNA-seq to correlate histone H4 occupancy with gene expression

    • ATAC-seq to relate histone H4 presence to chromatin accessibility

    • Mass spectrometry to identify histone H4 interaction partners

  • This integrated approach provides a comprehensive view of histone H4 function

• Disease-Specific Applications:

  • Cancer research: HIST1H4A (Ab-91) Antibody can be used to investigate histone onco-modifications in tumors

  • Neurodegenerative diseases: Study changes in histone H4 modifications in brain tissues

  • Developmental disorders: Examine histone H4 patterns during embryonic development

• DNA Damage Response Studies:

  • Build on findings that BBAP monoubiquitylates H4K91 to regulate the DNA damage response

  • Investigate how different DNA-damaging agents affect the balance between H4K91 modifications

  • Explore potential therapeutic approaches targeting these modifications

Recent research has revealed that histone H4 modifications function in coordinated networks rather than in isolation. The HIST1H4A (Ab-91) Antibody, in combination with modification-specific antibodies, enables researchers to decipher these complex regulatory networks.

What is the significance of HIST1H4A in the context of histone gene clusters and genomic organization?

Understanding the genomic context of HIST1H4A provides important insights for histone research:

• Genomic Organization:

  • HIST1H4A belongs to the HIST1 locus on chromosome 6p22.1-p22.2, which contains approximately 55 histone genes (about 80% of all histone genes)

  • Other histone H4 genes are distributed across different clusters: HIST2 and HIST3 on chromosome 1, and HIST4 on chromosome 12

  • This clustered organization facilitates coordinated expression during S phase

• Evolutionary Implications:

  • The high conservation of histone H4 across species reflects its fundamental role in chromatin organization

  • HIST1H4A (Ab-91) Antibody's cross-reactivity with human and mouse samples demonstrates this conservation

  • Comparative studies using this antibody can reveal species-specific aspects of histone regulation

• Replication-Dependent vs. Independent Expression:

  • Most histone genes in the HIST1 cluster, including HIST1H4A, are replication-dependent, with expression peaking during S phase

  • The HIST1H4A (Ab-91) Antibody can be used to study how replication-dependent histone expression is coordinated with DNA replication

  • ChIP experiments targeting DNA replication factors alongside HIST1H4A can reveal mechanisms of this coordination

• Transcriptional Regulation:

  • Transcription factors like C/EBPβ regulate histone H4 expression by binding to specific sites in the histone promoter

  • EMSA and ChIP experiments have identified C/EBP-binding sites in the histone H4 promoter, particularly in the region -125 to -117

  • This regulation is critical during processes like adipogenesis, where histone H4 expression facilitates mitotic clonal expansion

By considering HIST1H4A in its genomic context, researchers can design more comprehensive experiments that account for potential redundancy among histone genes and the coordinated regulation of histone expression.