HIST1H4A (Ab-20) Antibody

What is HIST1H4A and what role does it play in epigenetic research?

HIST1H4A (Histone Cluster 1, H4a) is one of several genes encoding the histone H4 protein, a core component of the nucleosome octamer around which DNA is wrapped in chromatin. Histone H4 plays a critical role in chromatin structure and gene regulation through various post-translational modifications (PTMs) at specific residues, particularly at lysine 20 (K20) .

The study of histone H4 modifications is central to epigenetic research as these modifications influence chromatin accessibility, transcriptional regulation, DNA damage response, and cell cycle progression. Specifically, modifications at lysine 20 (including methylation and acetylation) serve as important epigenetic markers associated with transcriptional repression, heterochromatin formation, and DNA repair mechanisms .

What does the "(Ab-20)" designation indicate in HIST1H4A antibodies?

The "(Ab-20)" designation in HIST1H4A antibodies refers to the specific epitope location targeted by the antibody - in this case, the antibody recognizes the region around lysine 20 (K20) of the histone H4 protein . This site-specific antibody was generated using a peptide sequence surrounding lysine 20 of human histone H4 as the immunogen .

This specificity is crucial for experimental design as K20 can undergo various modifications (methylation, acetylation) that have distinct biological implications. Researchers must carefully select the appropriate antibody based on whether they are investigating unmodified K20 or specific modifications like acetylation (acK20) or methylation (meK20) .

What applications has HIST1H4A (Ab-20) Antibody been validated for?

The HIST1H4A (Ab-20) Antibody has been validated for multiple research applications as evidenced by technical documentation:

This polyclonal antibody demonstrates consistent performance across these applications when used with human samples . For optimal results, researchers should perform dilution optimization experiments for their specific sample types and detection methods.

What is the host species and isotype of HIST1H4A (Ab-20) Antibody?

The HIST1H4A (Ab-20) Antibody is a rabbit-derived polyclonal antibody with IgG isotype . This information is important for several experimental considerations:

• Secondary antibody selection must be anti-rabbit IgG for detection systems

• When designing multiplex experiments, researchers must avoid using other rabbit-derived primary antibodies unless using specialized detection systems

• The polyclonal nature indicates multiple epitopes within the target region are recognized, which can enhance signal strength but may introduce background in some applications

How should researchers optimize Western blotting protocols for HIST1H4A (Ab-20) Antibody?

When performing Western blot analysis with HIST1H4A (Ab-20) Antibody, consider these specialized methodology recommendations:

• Sample preparation:

  • Use nuclear extraction protocols optimized for histone proteins

  • Include histone deacetylase inhibitors (e.g., sodium butyrate, trichostatin A) in lysis buffers to preserve acetylation status

  • Consider acid extraction methods for enriching histone fractions

• Gel electrophoresis conditions:

  • Use 15-18% SDS-PAGE gels to properly resolve the low molecular weight histone H4 (approximately 11 kDa)

  • Load appropriate controls including recombinant histone H4 with known modification status

• Transfer and detection optimization:

  • Use PVDF membranes with 0.2 μm pore size for better retention of small histone proteins

  • Optimize blocking conditions (3-5% BSA often performs better than milk for phospho-specific antibodies)

  • Perform overnight primary antibody incubation at 4°C at optimized dilution

  • Consider using signal enhancers for low abundance modifications

What are the critical considerations for ChIP experiments using HIST1H4A (Ab-20) Antibody?

Chromatin immunoprecipitation (ChIP) with HIST1H4A (Ab-20) Antibody requires careful optimization:

• Crosslinking optimization:

  • Standard formaldehyde crosslinking (1%) for 10 minutes is typically sufficient

  • For histone modification studies, some researchers prefer dual crosslinking with formaldehyde followed by ethylene glycol bis(succinimidyl succinate) (EGS)

• Chromatin fragmentation:

  • Sonication parameters should generate 200-500 bp fragments

  • Verify fragmentation efficiency by agarose gel electrophoresis before proceeding

• Antibody specificity controls:

  • Include IgG control from the same species (rabbit)

  • Consider peptide competition assays with modified and unmodified histone H4 peptides

  • Include genomic regions known to be enriched or depleted for H4K20 modifications

• Data analysis considerations:

  • Normalize to input DNA

  • Consider spike-in normalization with exogenous chromatin for quantitative comparisons

  • Validate findings with orthogonal approaches such as CUT&RUN or CUT&Tag

How can researchers verify antibody specificity for HIST1H4A K20 modifications?

Rigorous validation of antibody specificity is essential for histone modification studies:

• Peptide competition assays:

  • Pre-incubate the antibody with synthetic peptides containing the specific modification (e.g., H4K20ac)

  • Include controls with unmodified peptides and peptides with alternative modifications at the same site

  • A specific antibody signal should be competed away only by the correct modified peptide

• Dot blot analysis:

  • Spot serial dilutions of modified and unmodified peptides on membranes

  • Probe with the antibody to determine cross-reactivity

  • Similar to the approach shown in search result for the related H4K20me3 antibody

• Analysis in knockout/knockdown systems:

  • Use cells with knockout/knockdown of histone modifying enzymes specific for the modification

  • For example, knockdown of SETD8 (PR-Set7) should reduce H4K20me1 levels

  • This approach validates the antibody's ability to detect biologically relevant changes

• Mass spectrometry correlation:

  • Compare ChIP-seq or immunoblotting results with mass spectrometry quantification of histone modifications

  • This orthogonal approach provides additional confidence in antibody specificity

What optimization strategies are recommended for immunofluorescence with HIST1H4A (Ab-20) Antibody?

For optimal immunofluorescence results with HIST1H4A (Ab-20) Antibody:

• Fixation methods:

  • 4% paraformaldehyde fixation (10-15 minutes at room temperature) preserves most histone modifications

  • For certain modifications, methanol fixation (-20°C, 10 minutes) may provide better epitope accessibility

  • Critical: avoid overfixation which can mask epitopes

• Permeabilization optimization:

  • Use 0.1-0.3% Triton X-100 for nuclear permeabilization

  • Some protocols benefit from additional permeabilization with 0.1% SDS for 10 minutes

  • Test different permeabilization approaches for your specific cell type

• Dilution and incubation parameters:

  • Start with the recommended dilution range (1:20-1:200) and optimize

  • Extend primary antibody incubation to overnight at 4°C for weak signals

  • Consider signal amplification systems for low abundance modifications

• Controls and counterstaining:

  • Include secondary-only controls to assess background

  • Use DAPI or Hoechst for nuclear counterstaining

  • Consider co-staining with other nuclear markers for colocalization studies

What are the implications of different H4K20 modifications for data interpretation?

Understanding the biological significance of different H4K20 modifications is crucial for proper data interpretation:

• H4K20 monomethylation (H4K20me1):

  • Associated with transcriptional activation and elongation

  • Enriched at promoters of active genes

  • Cell cycle regulated, with highest levels during G2/M

  • Catalyzed primarily by SETD8/PR-Set7

• H4K20 di/trimethylation (H4K20me2/3):

  • Associated with transcriptional repression and heterochromatin formation

  • H4K20me3 is enriched at repetitive elements and imprinted genes

  • Catalyzed by SUV4-20H1/H2 enzymes

  • H4K20me3 signal in immunofluorescence often appears as distinct nuclear foci

• H4K20 acetylation (H4K20ac):

  • Less studied than methylation

  • Generally associated with active transcription

  • Often co-occurs with other active histone marks like H3K4me3

  • Dynamic mark regulated by specific histone acetyltransferases and deacetylases

• Crosstalk with other modifications:

  • H4K20 modifications can influence or be influenced by other nearby modifications

  • For example, H4K16ac and H4K20me3 tend to be mutually exclusive

  • Consider examining multiple modifications in parallel experiments

What are the most common causes of weak or absent signals when using HIST1H4A (Ab-20) Antibody?

When experiencing suboptimal signal with HIST1H4A (Ab-20) Antibody, investigate these potential causes:

• Epitope masking or destruction:

  • Overfixation can cross-link epitopes, making them inaccessible

  • Harsh extraction conditions may destroy modifications

  • Solution: Optimize fixation time and extract histones using gentler methods

• Low abundance of the modification:

  • H4K20 modifications vary significantly between cell types and conditions

  • Cell cycle phase influences H4K20 methylation states

  • Solution: Use positive control samples known to contain the modification of interest

• Antibody dilution issues:

  • Over-dilution leads to weak signals

  • Under-dilution may increase background

  • Solution: Perform a dilution series to determine optimal concentration

• Technical issues:

  • Buffer incompatibility (ensure glycerol percentage is appropriate)

  • Secondary antibody mismatch or degradation

  • Solution: Verify all reagents and use fresh working solutions

How should researchers validate experimental findings from HIST1H4A (Ab-20) Antibody studies?

Robust validation strategies enhance confidence in experimental findings:

• Technical validation:

  • Replicate experiments using independent biological samples

  • Use alternative antibody clones targeting the same modification

  • Employ complementary techniques (e.g., mass spectrometry)

• Biological validation:

  • Manipulate relevant histone-modifying enzymes (overexpression or knockdown)

  • Test samples with known differences in modification levels

  • Correlate with functional outcomes (e.g., gene expression changes)

• Quantitative considerations:

  • Use appropriate normalization strategies

  • For ChIP, normalize to input and total histone H4

  • For western blots, normalize to total H4 or other stable proteins

• Orthogonal approaches:

  • Compare ChIP-seq results with CUT&RUN or CUT&Tag

  • Validate immunofluorescence findings with biochemical fractionation

  • Use genetic approaches to alter modification levels

How can HIST1H4A (Ab-20) Antibody be integrated into multiplexed histone modification analysis?

For comprehensive epigenetic profiling, researchers can incorporate HIST1H4A (Ab-20) Antibody in multiplexed approaches:

• Sequential immunofluorescence:

  • Use primary antibodies from different host species

  • Alternatively, employ sequential staining with antibody stripping between rounds

  • Include co-localization analysis to identify genomic regions with overlapping modifications

• Multiparameter flow cytometry:

  • Fix and permeabilize cells using protocols optimized for nuclear antigens

  • Combine with antibodies against other modifications or proteins

  • Enables single-cell analysis of histone modification heterogeneity

• ChIP-re-ChIP approaches:

  • Perform sequential immunoprecipitations to identify regions with co-occurring modifications

  • First round: immunoprecipitate with HIST1H4A (Ab-20) Antibody

  • Second round: immunoprecipitate with antibody against another modification

  • This identifies genomic regions containing both modifications

• Mass cytometry (CyTOF):

  • Conjugate antibodies to metal isotopes

  • Allows simultaneous detection of numerous modifications

  • Provides single-cell resolution of the epigenetic landscape

What emerging technologies complement HIST1H4A (Ab-20) Antibody-based approaches?

Several cutting-edge technologies enhance or complement traditional antibody-based histone analysis:

• CUT&RUN and CUT&Tag:

  • More sensitive alternatives to ChIP with lower input requirements

  • Use targeted nuclease activity to release DNA fragments bound by the antibody

  • Compatible with HIST1H4A (Ab-20) Antibody with protocol modifications

• Single-cell epigenomics:

  • scChIP-seq, scCUT&Tag provide single-cell resolution of histone modifications

  • Reveals cellular heterogeneity in epigenetic states

  • Requires highly specific antibodies like HIST1H4A (Ab-20)

• Live-cell imaging of histone dynamics:

  • Combination of antibody fragments with cell-penetrating peptides

  • Allows visualization of histone modification dynamics in living cells

  • Provides temporal information not accessible with fixed-cell approaches

• Integrated multi-omics analysis:

  • Combine H4K20 modification mapping with transcriptomics, proteomics

  • Correlate modifications with functional outcomes

  • Computational integration of multiple data types enhances biological insights

How do cell cycle dynamics affect H4K20 modification patterns and experimental design?

H4K20 modifications exhibit strong cell cycle dependency, which must be considered in experimental design:

• Cell cycle variation of H4K20 methylation:

  • H4K20me1 peaks during G2/M phase (set by PR-Set7/SETD8)

  • H4K20me2/3 accumulate in G1 phase (catalyzed by SUV4-20H1/H2)

  • New histones deposited during S phase lack H4K20 methylation

• Experimental implications:

  • Synchronize cells when comparing H4K20 modification levels

  • For unsynchronized populations, consider cell cycle markers in multiparameter approaches

  • Single-cell approaches can resolve cell cycle-associated heterogeneity

• Biological significance:

  • H4K20me1 plays roles in mitotic chromosome condensation

  • H4K20me3 contributes to establishing repressive chromatin after DNA replication

  • Perturbations in these dynamics are associated with genomic instability

• Analytical considerations:

  • Include cell cycle stage markers in your analysis

  • Consider cell cycle arrest experiments to standardize modification patterns

  • Interpret results in the context of known cell cycle dynamics of H4K20 modifications