Acetyl-HIST1H1A (K21) Antibody

What is HIST1H1A and why is its K21 acetylation significant?

HIST1H1A (Histone H1.1) is a linker histone that binds to DNA between nucleosomes, forming the macromolecular structure known as the chromatin fiber. It plays a crucial role in the condensation of nucleosome chains into higher-order structured fibers . Acetylation at lysine 21 (K21) represents a key post-translational modification that reduces histone-DNA binding affinity, thereby promoting transcriptional activation. This specific modification is particularly important in epigenetic regulation of gene expression and chromatin accessibility, making it a valuable target for researchers studying chromatin dynamics and transcriptional regulation.

The significance of K21 acetylation extends beyond basic chromatin structure to functional consequences in cellular processes. When this site is acetylated, it alters the electrostatic interactions between the histone and DNA, contributing to a more relaxed chromatin state that facilitates the binding of transcription factors and other regulatory proteins. Understanding this modification provides insights into fundamental mechanisms of gene regulation.

What are the optimal applications for Acetyl-HIST1H1A (K21) Antibody?

The Acetyl-HIST1H1A (K21) Antibody has been validated for multiple research applications, primarily:

Each application provides different insights into acetylated HIST1H1A biology. ELISA offers quantitative measurement of acetylation levels, while microscopy-based techniques like IF and ICC allow visualization of nuclear distribution patterns and co-localization with other nuclear proteins. The antibody has been specifically validated in human cells, with particular success in HeLa cells treated with histone deacetylase inhibitors like sodium butyrate .

How should the antibody be stored and handled to maintain optimal activity?

Proper storage and handling are critical for maintaining antibody activity and specificity:

• Storage Temperature: Store at -20°C or -80°C upon receipt .

• Buffer Composition: The antibody is provided in a buffer containing 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4 .

• Aliquoting: To minimize freeze-thaw cycles, divide the antibody into small working aliquots before freezing.

• Thawing Protocol: Thaw on ice and centrifuge briefly before use to collect contents at the bottom of the tube.

• Freeze-Thaw Cycles: Avoid repeated freeze-thaw cycles as they can degrade antibody quality and reduce specificity .

For long-term experiments, it's advisable to prepare multiple small aliquots during initial thawing to minimize repeated freezing. When diluting for specific applications, use fresh, sterile buffers and prepare dilutions immediately before use rather than storing diluted antibody for extended periods.

What controls should be included when using this antibody?

When designing experiments with the Acetyl-HIST1H1A (K21) Antibody, the following controls are recommended:

• Positive Control: HeLa cells treated with 30mM sodium butyrate for 4 hours have been validated as an effective positive control . This treatment inhibits histone deacetylases, resulting in hyperacetylation of histones including HIST1H1A at K21.

• Negative Controls:

  • Untreated cells (without HDAC inhibitors) to establish baseline acetylation levels

  • Secondary antibody-only control to assess non-specific binding

  • Isotype control (rabbit IgG) at the same concentration as the primary antibody

• Specificity Controls:

  • Peptide competition assay using the immunizing peptide (amino acids 18-30 of Histone H1.1)

  • HIST1H1A knockdown/knockout cells to confirm signal specificity

These controls help distinguish genuine acetyl-HIST1H1A signal from background and ensure experimental reliability. The peptide competition assay is particularly valuable for confirming epitope-specific binding.

How does acetylation at K21 affect HIST1H1A function in chromatin dynamics?

Acetylation at K21 of HIST1H1A significantly impacts chromatin structure and dynamics through several mechanisms:

• Reduced DNA Binding Affinity: K21 acetylation neutralizes the positive charge of lysine, weakening electrostatic interactions between HIST1H1A and the negatively charged DNA backbone. This reduction in binding affinity leads to a more dynamic association of H1.1 with chromatin.

• Altered Chromatin Compaction: Modified HIST1H1A has decreased capability to promote higher-order chromatin folding, resulting in more accessible chromatin regions. This structural change affects nucleosome spacing and positioning.

• Transcriptional Regulation: Regions with acetylated HIST1H1A show increased gene expression due to:

  • Enhanced accessibility for transcription factors

  • Altered recruitment of chromatin remodeling complexes

  • Modified interactions with other histone modifications

• Cell Cycle Regulation: HIST1H1A acetylation patterns change throughout the cell cycle, with evidence suggesting coordination between K21 acetylation and other post-translational modifications to regulate replication timing and mitotic progression.

Experimentally, these effects can be observed through techniques like FRAP (Fluorescence Recovery After Photobleaching), which demonstrates increased mobility of acetylated H1.1 compared to unmodified forms.

What experimental protocols optimize detection of acetylated HIST1H1A in immunocytochemistry?

For optimal immunocytochemistry results with Acetyl-HIST1H1A (K21) Antibody, the following validated protocol has shown excellent results:

• Cell Preparation:

  • Culture cells on appropriate coverslips or chamber slides

  • For enhanced signal, treat cells with HDAC inhibitors (e.g., 30mM sodium butyrate for 4 hours)

• Fixation and Permeabilization:

  • Fix cells in 4% formaldehyde for 15 minutes at room temperature

  • Permeabilize using 0.2% Triton X-100 for 10 minutes

• Blocking:

  • Block with 10% normal goat serum for 30 minutes at room temperature

• Primary Antibody Incubation:

  • Dilute Acetyl-HIST1H1A (K21) Antibody at 1:25 (optimal starting dilution)

  • Prepare antibody in 1% BSA solution

  • Incubate overnight at 4°C

• Detection:

  • Use biotinylated secondary antibody followed by HRP-conjugated streptavidin-peroxidase system

  • Alternatively, fluorescently-labeled secondary antibodies can be used for co-localization studies

• Counterstaining:

  • DAPI for nuclear visualization

  • Consider co-staining with other histone marks to study modification patterns

• Image Acquisition:

  • Use confocal microscopy for detailed nuclear localization patterns

  • Employ deconvolution techniques for improved resolution of subnuclear structures

This protocol has been validated on a Leica Bond™ system, but can be adapted for standard immunocytochemistry workflows with proper optimization .

How can researchers distinguish between acetylation at K21 and other post-translational modifications of HIST1H1A?

Distinguishing between acetylation at K21 and other post-translational modifications (PTMs) of HIST1H1A requires careful experimental design:

• Antibody Validation Techniques:

  • Peptide array analysis with modified and unmodified peptides

  • Western blotting with multiple antibodies against different HIST1H1A modifications

  • Mass spectrometry validation of modification-specific signals

• Potential Cross-Reactivity Concerns:

  • Acetylation specificity may be affected by adjacent modifications (phosphorylation, methylation)

  • Antibody performance should be validated when studying cells with multiple simultaneous modifications

• Modification-Specific Approaches:

  • Use site-specific mutants (K21R) to confirm antibody specificity

  • Employ HDAC inhibitors of different classes to distinguish between modification sites

  • Combine with mass spectrometry for comprehensive PTM profiling

• Technical Considerations:

  • Adjacent modifications can sterically hinder antibody binding

  • Modification crosstalk may affect epitope accessibility

When designing experiments to study K21 acetylation specifically, researchers should consider the "modification neighborhood" and employ complementary approaches like mass spectrometry to confirm antibody-based findings.

What is the relationship between HIST1H1A K21 acetylation and cancer progression?

Research indicates several important connections between HIST1H1A K21 acetylation and cancer:

• Altered Acetylation Patterns:

  • Aberrant histone acetylation, including HIST1H1A K21, has been documented in breast and lung adenocarcinoma

  • These changes correlate with dysregulated gene expression profiles in cancer cells

• Oncogenic Pathway Association:

  • Elevated acetylation of linker histones correlates with activation of oncogenic pathways:

    • TGFβ signaling pathway activation

    • SOX9 transcription factor upregulation

• Diagnostic and Prognostic Potential:

  • Modified HIST1H1A patterns may serve as biomarkers for certain cancer subtypes

  • Changes in K21 acetylation correlate with altered chromatin accessibility at cancer-associated genes

• Therapeutic Implications:

  • HDAC inhibitors, which increase histone acetylation including at HIST1H1A K21, show promising anticancer activity

  • Understanding the specific role of K21 acetylation may help optimize epigenetic therapies

When investigating cancer connections, researchers should consider examining:

• Acetylation levels in matched tumor/normal tissue pairs

• Correlation between K21 acetylation and expression of key oncogenes

• Changes in acetylation patterns during cancer progression

How can ChIP-seq be optimized for studying genome-wide binding patterns of acetylated HIST1H1A?

Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) with Acetyl-HIST1H1A (K21) Antibody requires specific optimization:

• Crosslinking and Chromatin Preparation:

  • Optimize formaldehyde crosslinking time (typically 10-15 minutes)

  • Use sonication conditions that generate 200-500bp fragments

  • Consider dual crosslinking (formaldehyde + DSG) for improved histone-DNA interactions

• Immunoprecipitation Optimization:

  • Antibody amount: Start with 2-5μg per IP reaction

  • Increase antibody:chromatin ratio compared to core histone ChIP

  • Extended incubation time (overnight at 4°C) for maximal binding

• Controls and Validation:

  • Input DNA control

  • IgG control to assess background

  • Spike-in of exogenous chromatin for normalization

  • qPCR validation at known targets before sequencing

• Bioinformatic Analysis Considerations:

  • Use peak calling algorithms optimized for histone modifications (e.g., MACS2 with broad peak settings)

  • Compare acetylated HIST1H1A binding with:

    • Transcriptionally active regions

    • Other histone modifications (H3K27ac, H3K4me3)

    • Chromatin accessibility data (ATAC-seq, DNase-seq)

• Experimental Design Recommendations:

  • Include treatments that modulate acetylation (HDAC inhibitors, HAT inhibitors)

  • Consider cell cycle synchronization to capture dynamic binding

  • Perform replicate experiments for statistical robustness

This approach enables genome-wide profiling of acetylated HIST1H1A distribution and its relationship to chromatin states and gene regulation.

Sample validation data for Acetyl-HIST1H1A (K21) Antibody

The following data summarizes key validation experiments performed with this antibody:

These data demonstrate the specificity and utility of the antibody across multiple experimental approaches. The enhancement of signal with HDAC inhibitor treatment confirms the acetylation-specific nature of the antibody.

Technical troubleshooting for common issues

This troubleshooting guide addresses the most common challenges researchers face when working with histone modification-specific antibodies.