HIST1H4A (Ab-88) Antibody

What is HIST1H4A (Ab-88) Antibody and what epitope does it recognize?

HIST1H4A (Ab-88) Antibody is a specialized antibody designed to recognize histone H4 when phosphorylated at tyrosine 88 (pY88-H4). This antibody specifically targets this post-translational modification with high affinity and specificity. Histone H4 is a core component of nucleosomes that wrap and compact DNA into chromatin, playing a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability .

The antibody has been validated to not cross-react with unphosphorylated H4 peptides or with other histone modifications, making it a valuable tool for studying this specific post-translational modification . Validation studies have confirmed its specificity through peptide competition assays, where phosphopeptides competed with pY88-H4 antibody for binding, dampening the signal .

What are the validated applications for HIST1H4A (Ab-88) Antibody?

The HIST1H4A (Ab-88) Antibody has been validated for multiple experimental applications:

These applications allow researchers to investigate the role of H4 Tyr88 phosphorylation in various biological contexts, including gene regulation and disease mechanisms .

How has specificity of HIST1H4A (Ab-88) Antibody been validated?

The specificity of HIST1H4A (Ab-88) Antibody has been rigorously validated through multiple approaches:

• Peptide recognition testing: The antibody specifically recognizes Tyr88-phosphorylated H4 peptide but fails to recognize unphosphorylated peptides .

• Cross-reactivity screening: The antibody was tested against 59 different histone modifications (including acetylation, methylation, phosphorylation, and citrullination) and showed no cross-reactivity .

• Mutational analysis: Point mutation studies confirmed the antibody recognizes the Y88 phosphorylation site specifically, as it fails to recognize Y88F-H4 mutants (where tyrosine 88 is replaced with phenylalanine) while still recognizing Y72F-H4 mutants .

• Kinase inhibition: Treatment with ACK1 inhibitors like (R)-9bMS eliminated the pY88-H4 signal, confirming the specificity of the antibody for this phosphorylation event .

• Genetic validation: Knockdown of ACK1 expression using siRNA or CRISPR-Cas9 gene editing resulted in significant loss of H4 Y88-phosphorylation detection .

These comprehensive validation approaches ensure that HIST1H4A (Ab-88) Antibody provides reliable and specific detection of pY88-H4.

What biological significance does Tyr88 phosphorylation on histone H4 have?

Tyrosine 88 phosphorylation on histone H4 represents a specific post-translational modification with important biological functions:

• Gene regulation: ChIP-sequencing studies have revealed that pY88-H4 marks are deposited at approximately 370 distinct genomic locations, including three sites upstream of the androgen receptor (AR) gene transcription start site .

• Cancer biology: pY88-H4 has been implicated in the regulation of AR gene expression in prostate cancer, particularly in castration-resistant prostate cancer (CRPC) where AR signaling remains active despite androgen deprivation therapy .

• Signaling pathway integration: H4 Tyr88 phosphorylation is regulated by the tyrosine kinase ACK1 (TNK2), which responds to growth factor signaling such as IGF stimulation .

• Epigenetic programming: As a histone modification, pY88-H4 contributes to the "histone code" that regulates DNA accessibility and gene expression patterns .

Understanding this modification provides insights into epigenetic mechanisms of gene regulation and potential therapeutic targets for diseases where these pathways are dysregulated.

What are the optimal validation methods to confirm HIST1H4A (Ab-88) Antibody specificity in new experimental systems?

When implementing HIST1H4A (Ab-88) Antibody in a new experimental system, researchers should perform a comprehensive validation protocol:

• Peptide competition assays: Pre-incubate the antibody with phosphorylated and non-phosphorylated peptides separately. The phosphorylated peptide should compete for binding and reduce signal, while the non-phosphorylated peptide should not affect binding .

• Genetic approaches:

  • Express wild-type H4 and Y88F-H4 mutant constructs to demonstrate specificity

  • Perform ACK1 knockdown/knockout experiments using siRNA or CRISPR-Cas9

• Pharmacological validation:

  • Treat cells with ACK1 inhibitors like (R)-9bMS to reduce pY88-H4 levels

  • Stimulate cells with growth factors (e.g., IGF) to increase pY88-H4 levels

• Signal characteristics verification:

  • Confirm nuclear localization in immunofluorescence experiments

  • Verify the expected molecular weight (11-14 kDa) in Western blots

• Orthogonal detection methods:

  • Confirm findings using mass spectrometry or alternative antibody clones if available

This multi-faceted validation approach ensures reliable detection of pY88-H4 in any experimental system.

How should ChIP-sequencing experiments using HIST1H4A (Ab-88) Antibody be designed?

Successful ChIP-sequencing with HIST1H4A (Ab-88) Antibody requires careful experimental design:

• Chromatin preparation:

  • Optimize crosslinking conditions (typically 1% formaldehyde for 10 minutes)

  • Determine optimal sonication parameters to generate 200-500 bp fragments

  • Include phosphatase inhibitors throughout to preserve pY88-H4

• Immunoprecipitation protocol:

  • Determine optimal antibody concentration through titration experiments

  • Include appropriate controls:

    • Input chromatin (non-immunoprecipitated)

    • IgG control (non-specific antibody of the same isotype)

    • Known positive control regions (e.g., AR gene regulatory elements)

• Library preparation and sequencing:

  • Generate sufficient sequencing depth (minimum 20 million uniquely mapped reads)

  • Include biological replicates (minimum n=3) for statistical robustness

• Data analysis pipeline:

  • Use appropriate peak calling algorithms (e.g., MACS2)

  • Apply normalization methods accounting for input and IgG controls

  • Perform genome browser visualization and motif analysis

• Validation of key findings:

  • Confirm selected peak regions by ChIP-qPCR

  • Correlate with gene expression data where relevant

Previous ChIP-seq studies using pY88-H4 antibody successfully identified approximately 370 distinct genomic locations where this mark is deposited, demonstrating the feasibility of this approach .

What factors affect the stability and detection of pY88-H4 during sample preparation?

The phosphorylation of histone H4 at Tyr88 can be labile during sample preparation, requiring specific precautions:

• Phosphatase inhibitors:

  • Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride, phosphatase inhibitor cocktails) in all buffers

  • Add inhibitors fresh before each experiment

• Temperature considerations:

  • Maintain samples at 4°C during processing to minimize enzymatic activity

  • Avoid extended incubations at room temperature

• Buffer composition:

  • Use buffers with neutral to slightly alkaline pH (7.4-8.0)

  • Include detergents appropriate for nuclear protein extraction (e.g., NP-40, Triton X-100)

• Fixation methods:

  • For immunofluorescence: 4% paraformaldehyde for 10-15 minutes

  • For ChIP: 1% formaldehyde for 10 minutes

  • Over-fixation can mask epitopes while under-fixation risks epitope loss

• Protein denaturation:

  • For Western blotting, use SDS-PAGE with reducing conditions

  • Avoid excessive heating of samples (65°C for 5-10 minutes preferred over boiling)

• Storage considerations:

  • Process samples immediately when possible

  • For storage, flash-freeze in liquid nitrogen and maintain at -80°C

  • Minimize freeze-thaw cycles

• Blocking reagents:

  • Use BSA (1-5%) rather than milk for immunodetection, as milk contains phosphoproteins that may interfere with detection

Careful attention to these factors helps preserve pY88-H4 during sample preparation and ensures optimal detection.

How can multi-parameter analysis of histone modifications including pY88-H4 be performed?

Combining HIST1H4A (Ab-88) Antibody with detection of other histone modifications enables comprehensive epigenetic profiling:

• Sequential ChIP (Re-ChIP):

  • First immunoprecipitation with HIST1H4A (Ab-88) Antibody

  • Elution under mild conditions

  • Second immunoprecipitation with antibody against another modification

  • Analysis reveals regions where both modifications co-exist

• Multiplex immunofluorescence:

  • Use antibodies from different host species (e.g., rabbit anti-pY88-H4 with mouse anti-H3K4me3)

  • Apply species-specific secondary antibodies with distinct fluorophores

  • Include proper controls for antibody cross-reactivity

  • Analysis by confocal microscopy or high-content imaging

• Mass spectrometry approaches:

  • Implement amino acid coded mass tagging (AACM) as described for histone analysis

  • Digest histones with appropriate proteases (e.g., trypsin, Arg-C)

  • Analyze peptides by MALDI-TOF MS or LC-MS/MS

  • Quantify relative abundance of different modifications

• Integrative genomics:

  • Perform parallel ChIP-seq experiments with different modification-specific antibodies

  • Integrate datasets to identify patterns of co-occurrence or mutual exclusivity

  • Correlate with transcriptomic data (RNA-seq) to determine functional outcomes

These approaches provide a comprehensive view of how pY88-H4 functions within the broader context of histone modifications and chromatin regulation.

What troubleshooting strategies should be employed when HIST1H4A (Ab-88) Antibody produces suboptimal results?

When encountering difficulties with HIST1H4A (Ab-88) Antibody, systematic troubleshooting is essential:

• Low or no signal:

  • Verify phosphorylation status: Confirm ACK1 activity in your experimental system

  • Stimulate with growth factors (e.g., IGF) to increase pY88-H4 levels

  • Optimize antibody concentration: Try a range around the recommended dilution (1:50-1:500)

  • Improve epitope accessibility: Test different fixation/permeabilization conditions

  • Check antibody storage: Ensure proper storage at -20°C and minimal freeze-thaw cycles

• High background or non-specific signals:

  • Increase blocking stringency: Use 3-5% BSA and extend blocking time

  • Optimize antibody dilution: Too concentrated antibody can increase background

  • Increase wash stringency: More washes or higher salt concentration

  • Perform peptide competition assays to distinguish specific from non-specific signals

  • Include phosphatase inhibitors to prevent loss of phosphorylation

• Inconsistent results between experiments:

  • Standardize cell culture conditions: Control cell density, passage number

  • Maintain consistent stimulation protocols: Time, concentration of stimulants

  • Use the same lot of antibody when possible

  • Include positive and negative controls in each experiment

  • Maintain strict timing for all protocol steps

• Unexpected molecular weight or localization:

  • Verify antibody specificity using mutants (Y88F-H4)

  • Confirm with orthogonal methods (mass spectrometry)

  • Check for post-translational modification crosstalk affecting epitope recognition

  • Ensure complete protein denaturation for Western blotting

A methodical approach to troubleshooting can help identify and resolve issues when working with HIST1H4A (Ab-88) Antibody.

How should quantitative analysis of pY88-H4 signals be performed?

Accurate quantification of pY88-H4 signals requires appropriate normalization and analytical approaches:

• Western blot quantification:

  • Normalize pY88-H4 signal to total H4 from the same samples

  • Use linear range of detection (avoid saturated signals)

  • Include a standard curve of known quantities when possible

  • Apply densitometry software with background subtraction

• Immunofluorescence quantification:

  • Measure nuclear intensity relative to background

  • Normalize to DAPI or total H4 staining

  • Analyze sufficient cell numbers (>100 cells per condition)

  • Consider single-cell analysis to capture population heterogeneity

• ChIP-qPCR quantification:

  • Express as percent of input (% input)

  • Calculate fold enrichment over IgG control

  • Include positive and negative control regions

  • Normalize to a housekeeping gene lacking pY88-H4 modification

• Flow cytometry analysis:

  • Report median fluorescence intensity (MFI) rather than mean values

  • Use appropriate gating strategies based on controls

  • Present data as fold change relative to control conditions

• Statistical analysis:

  • Perform experiments with sufficient biological replicates (n≥3)

  • Apply appropriate statistical tests based on data distribution

  • Report p-values and confidence intervals

  • Consider multiple testing corrections for genome-wide analyses

These quantification approaches ensure accurate and reproducible analysis of pY88-H4 signals across different experimental platforms.