Acetyl-HIST1H2AG (K9) Antibody

What is HIST1H2AG protein and what is its biological function?

HIST1H2AG, also known as Histone H2A type 1 (H2A.1) or Histone H2A/ptl, is a core component of nucleosomes, which are fundamental units of chromatin structure. As a core histone protein, HIST1H2AG plays a central role in several critical cellular processes including:

• Compaction of DNA into chromatin, limiting DNA accessibility to cellular machinery

• Regulation of transcription through modulation of DNA accessibility

• Participation in DNA repair mechanisms

• Ensuring proper DNA replication

• Maintenance of chromosomal stability

The nucleosome consists of approximately 146bp of DNA wrapped around a histone octamer composed of four major histone types: H2A, H2B, H3, and H4. HIST1H2AG specifically contributes to proper centromere assembly and function, which is essential for chromosome segregation during cell division .

What does the "K9" in Acetyl-HIST1H2AG (K9) Antibody refer to?

The "K9" in Acetyl-HIST1H2AG (K9) Antibody refers to the specific lysine residue (the 9th lysine) in the HIST1H2AG protein sequence that has been acetylated. This post-translational modification occurs when an acetyl group is added to the ε-amino group of the lysine side chain at position 9.

The antibody is specifically designed to recognize and bind to HIST1H2AG protein only when this particular lysine residue is acetylated, making it a valuable tool for studying this specific epigenetic modification . Similar antibodies exist for other acetylation sites on the same protein, such as K15, K36, and K74, each targeting different lysine residues that can undergo acetylation .

What applications is the Acetyl-HIST1H2AG (K9) Antibody suitable for?

Based on manufacturer specifications, the Acetyl-HIST1H2AG (K9) Antibody is validated for the following applications:

The antibody can be used to investigate the presence and distribution of acetylated HIST1H2AG (K9) in various experimental contexts, including:

• Studying changes in histone acetylation during cellular processes

• Examining the effect of histone deacetylase inhibitors

• Investigating epigenetic patterns in different cell types or disease states

While not explicitly mentioned in the search results for the K9 variant, similar antibodies targeting other acetylation sites on HIST1H2AG are also compatible with Western Blotting (WB) and Immunofluorescence (IF) applications .

How does acetylation at K9 of HIST1H2AG differ functionally from other acetylation sites?

Histone H2A type 1 (HIST1H2AG) can be acetylated at multiple lysine residues, including K9, K15, K36, and K74, each with potentially distinct functional implications for chromatin regulation.

K9 acetylation on HIST1H2AG is generally associated with active transcription and is often found in promoter regions of actively transcribed genes. In contrast:

• K15 acetylation may be involved in DNA damage response pathways and is often modified during DNA repair processes

• K36 acetylation is frequently associated with transcriptional elongation and may serve as a mark for recruiting specific transcriptional complexes

• K74 acetylation is less well-characterized but may play roles in chromatin remodeling during specific cellular processes

Understanding these site-specific differences is crucial for interpreting experimental results when using acetylation site-specific antibodies in epigenetic research.

What are the recommended protocols for using Acetyl-HIST1H2AG (K9) Antibody in chromatin immunoprecipitation (ChIP) assays?

While ChIP is not specifically listed among the validated applications for this antibody in the search results, the following protocol can be adapted based on general principles for histone modification ChIP assays and the properties of this antibody:

Optimized ChIP Protocol for Acetyl-HIST1H2AG (K9) Antibody:

• Cell Fixation and Chromatin Preparation:

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

  • Quench with 125 mM glycine for 5 minutes

  • Lyse cells and isolate nuclei using appropriate buffers

  • Sonicate chromatin to fragments of 200-500 bp

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate 2-5 μg of Acetyl-HIST1H2AG (K9) antibody with chromatin overnight at 4°C

  • Add protein A/G beads and incubate for 2-4 hours

  • Wash extensively with low and high salt buffers

• DNA Recovery and Analysis:

  • Reverse crosslinks at 65°C overnight

  • Treat with RNase A and Proteinase K

  • Purify DNA using column-based methods

  • Analyze by qPCR, sequencing, or other downstream applications

Critical Controls:

• Input chromatin (non-immunoprecipitated)

• IgG negative control

• Positive control using antibody against a known abundant histone mark (e.g., H3K4me3)

• Genomic regions known to be enriched or depleted for H2A acetylation

Successful ChIP experiments typically require optimization of antibody concentration, chromatin amount, and incubation conditions for each specific cell type and experimental condition.

How can I investigate the interplay between HIST1H2AG K9 acetylation and other histone modifications?

Investigating the interplay between different histone modifications requires sophisticated experimental approaches. Here are methodological approaches for such studies:

• Sequential ChIP (Re-ChIP):

  • Perform first immunoprecipitation with Acetyl-HIST1H2AG (K9) antibody

  • Elute the chromatin complexes

  • Perform second immunoprecipitation with antibody against another histone modification

  • This identifies genomic regions where both modifications co-exist

• Mass Spectrometry Analysis:

  • Immunoprecipitate histones using Acetyl-HIST1H2AG (K9) antibody

  • Perform proteomic analysis to identify co-occurring modifications

  • Quantify relative abundance of different modification patterns

• Multiplexed Immunofluorescence:

  • Use Acetyl-HIST1H2AG (K9) antibody alongside antibodies against other histone marks

  • Employ different fluorophores for each antibody

  • Analyze co-localization patterns using confocal microscopy

• Combined ChIP-seq and RNA-seq:

  • Perform ChIP-seq with Acetyl-HIST1H2AG (K9) antibody

  • Conduct parallel RNA-seq from the same samples

  • Correlate acetylation patterns with gene expression profiles

  • Compare with published datasets for other histone modifications

These approaches can reveal whether K9 acetylation of HIST1H2AG acts synergistically or antagonistically with other histone modifications in regulating chromatin structure and gene expression.

What are the optimal fixation and permeabilization protocols for detecting Acetyl-HIST1H2AG (K9) in immunocytochemistry?

For optimal detection of Acetyl-HIST1H2AG (K9) in immunocytochemistry applications, the following protocol is recommended based on general practices for histone modification detection:

Fixation Protocol:

• Grow cells on coverslips to 70-80% confluence

• Rinse cells gently with pre-warmed PBS

• Fix with 4% paraformaldehyde in PBS for 15 minutes at room temperature

• Wash three times with PBS, 5 minutes each

Permeabilization Protocol:

• Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes at room temperature

• Wash three times with PBS, 5 minutes each

• Block with 5% normal serum (from the same species as the secondary antibody) in PBS for 1 hour at room temperature

Immunostaining:

• Incubate with Acetyl-HIST1H2AG (K9) antibody diluted 1:10 to 1:100 in blocking buffer overnight at 4°C

• Wash three times with PBS, 5 minutes each

• Incubate with fluorophore-conjugated secondary antibody for 1 hour at room temperature

• Wash three times with PBS, 5 minutes each

• Counterstain nuclei with DAPI

• Mount and observe using appropriate microscopy

Critical Notes:

• Avoid excessive washing or harsh permeabilization that may extract nuclear proteins

• Include appropriate controls (omission of primary antibody, peptide competition)

• For dual staining with other nuclear markers, optimize antibody combinations carefully to avoid cross-reactivity

How should I validate the specificity of the Acetyl-HIST1H2AG (K9) Antibody?

Validating antibody specificity is crucial for reliable experimental results. Here are comprehensive approaches to validate the Acetyl-HIST1H2AG (K9) antibody:

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess acetylated K9 peptide

  • Use this mixture in parallel with the normal antibody

  • Signal should be significantly reduced or eliminated with the peptide-blocked antibody

• Knockout/Knockdown Controls:

  • Use CRISPR/Cas9 to knockout HIST1H2AG or siRNA to knockdown its expression

  • Compare signal between wild-type and knockout/knockdown samples

  • Signal should be reduced or absent in knockout/knockdown samples

• Deacetylase Treatment:

  • Treat samples with histone deacetylases (HDACs) to remove acetyl groups

  • Compare signal before and after treatment

  • Signal should be reduced after HDAC treatment

• Multiple Antibody Validation:

  • Test multiple antibodies against the same epitope from different suppliers

  • Compare staining patterns and signal intensities

  • Consistent results across different antibodies increase confidence in specificity

• Western Blot Analysis:

  • Run histone extracts from different cell types or treatments

  • Probe with Acetyl-HIST1H2AG (K9) antibody

  • Confirm single band at the expected molecular weight (~14 kDa)

  • Test with acetylated and non-acetylated recombinant HIST1H2AG proteins

• Mass Spectrometry Confirmation:

  • Immunoprecipitate with the antibody

  • Analyze immunoprecipitated proteins by mass spectrometry

  • Confirm presence of acetylated K9 peptides from HIST1H2AG

This multi-faceted validation approach ensures that your experimental results truly reflect the biological status of K9 acetylation on HIST1H2AG.

What factors affect the sensitivity and reproducibility of experiments using Acetyl-HIST1H2AG (K9) Antibody?

Several factors can influence the sensitivity and reproducibility of experiments using histone modification antibodies like Acetyl-HIST1H2AG (K9) Antibody:

Technical Factors:

Biological Factors:

By carefully controlling these factors, researchers can enhance the reproducibility and reliability of experiments using the Acetyl-HIST1H2AG (K9) Antibody.

How can Acetyl-HIST1H2AG (K9) Antibody be used in studying epigenetic changes during cellular differentiation?

The Acetyl-HIST1H2AG (K9) Antibody can be a powerful tool for investigating epigenetic reprogramming during cellular differentiation. Here's a comprehensive approach:

• Time-Course Analysis:

  • Collect cells at defined stages of differentiation

  • Perform immunofluorescence or ChIP-seq with Acetyl-HIST1H2AG (K9) antibody

  • Map dynamic changes in K9 acetylation patterns

  • Correlate with expression of lineage-specific genes

• Comparative Profiling Across Lineages:

  • Apply the antibody to cells differentiating along different lineages

  • Identify common and lineage-specific K9 acetylation changes

  • Create genome-wide maps of acetylation dynamics

• Integration with Transcription Factor Binding:

  • Combine ChIP-seq for Acetyl-HIST1H2AG (K9) with ChIP-seq for lineage-determining transcription factors

  • Identify genomic regions where K9 acetylation precedes, coincides with, or follows transcription factor binding

  • Determine causal relationships in epigenetic reprogramming

• Perturbation Studies:

  • Inhibit histone acetyltransferases (HATs) or deacetylases (HDACs)

  • Assess impact on differentiation trajectory

  • Use Acetyl-HIST1H2AG (K9) antibody to monitor resulting changes in acetylation patterns

  • Correlate with altered differentiation outcomes

• Single-Cell Applications:

  • Adapt protocols for single-cell immunofluorescence or CUT&Tag

  • Map heterogeneity in acetylation patterns within differentiating populations

  • Identify cells with pioneer acetylation events that precede fate commitment

This multifaceted approach can reveal how K9 acetylation on HIST1H2AG contributes to establishing and maintaining cell fate decisions during development and differentiation.

What are the best practices for multiplexed detection of histone modifications including Acetyl-HIST1H2AG (K9)?

Multiplexed detection of histone modifications enables researchers to study the combinatorial epigenetic code. Here are best practices for including Acetyl-HIST1H2AG (K9) in multiplexed studies:

For Immunofluorescence/Immunocytochemistry:

• Antibody Selection:

  • Choose primary antibodies raised in different host species (e.g., rabbit anti-Acetyl-HIST1H2AG (K9) paired with mouse anti-H3K27me3)

  • Confirm specificity of each antibody independently before multiplexing

  • Ensure that cross-reactivity testing has been performed

• Sequential Staining Protocol:

  • For antibodies from the same species, use sequential staining with intermediate blocking

  • First primary antibody → first secondary antibody → blocking → second primary antibody → second secondary antibody

  • Use appropriate controls to ensure signal specificity

• Fluorophore Selection:

  • Choose fluorophores with minimal spectral overlap

  • Consider brightness when designing panels (brighter fluorophores for less abundant marks)

  • Account for autofluorescence of the sample

• Image Acquisition and Analysis:

  • Collect single-color controls for spectral unmixing

  • Use consistent exposure settings between experiments

  • Employ quantitative image analysis software for co-localization studies

For Mass Cytometry/CyTOF Applications:

• Metal-Conjugated Antibody Preparation:

  • Conjugate Acetyl-HIST1H2AG (K9) antibody with a specific metal isotope

  • Titrate antibody concentration for optimal signal-to-noise ratio

  • Include isotype controls for background assessment

• Sample Processing:

  • Optimize cell fixation and permeabilization for nuclear epitope access

  • Consider using cell barcoding for batch processing

  • Include spike-in controls for technical normalization

• Panel Design:

  • Include relevant histone marks that may correlate or anti-correlate with K9 acetylation

  • Add cell cycle markers to account for cell cycle-dependent variations

  • Include markers for cell identity in heterogeneous samples

These approaches allow for comprehensive characterization of the epigenetic landscape, revealing how Acetyl-HIST1H2AG (K9) interacts with other histone modifications in regulating chromatin structure and gene expression.

How can I troubleshoot weak or absent signal when using Acetyl-HIST1H2AG (K9) Antibody?

If you encounter weak or absent signal when using the Acetyl-HIST1H2AG (K9) Antibody, consider the following troubleshooting approaches:

Primary Causes and Solutions:

Verification Steps:

• Perform Western blot to confirm presence of acetylated HIST1H2AG in your samples

• Use positive control cell lines known to have high levels of histone acetylation

• Run parallel experiments with antibodies against different acetylation sites

• Confirm antibody activity using dot blot with acetylated peptides

By systematically addressing these potential issues, you can optimize detection of Acetyl-HIST1H2AG (K9) in your experimental system.

What quality control parameters should be monitored when using Acetyl-HIST1H2AG (K9) Antibody?

To ensure reliable and reproducible results with Acetyl-HIST1H2AG (K9) Antibody, the following quality control parameters should be regularly monitored:

Antibody Quality Control:

• Antibody Performance Tracking:

  • Maintain control cell lysates with known levels of K9 acetylation

  • Periodically test antibody performance against these standards

  • Document signal intensity and specificity over time to detect degradation

• Batch Consistency:

  • Record lot numbers and maintain records of performance

  • When changing lots, perform side-by-side comparison with previous lot

  • Document any variations in sensitivity or specificity

Experimental Quality Control:

• Positive and Negative Controls:

  • Include HDAC inhibitor-treated cells as positive control

  • Use HDAC-overexpressing cells as negative control

  • Include isotype control antibody to assess background

• Signal-to-Noise Ratio:

  • Calculate and track signal-to-background ratios

  • Maintain consistent imaging or detection parameters

  • Establish minimum acceptable threshold for signal-to-noise ratio

• Reproducibility Assessment:

  • Periodically repeat key experiments to ensure consistent results

  • Perform technical replicates with different personnel if possible

  • Document inter-experiment variability

• Cross-Reactivity Monitoring:

  • Test against samples with various histone modification patterns

  • Verify specificity using peptide competition assays

  • Confirm absence of signal in HIST1H2AG knockout/knockdown samples

Implementing these quality control measures will help ensure that experimental results obtained with Acetyl-HIST1H2AG (K9) Antibody are reliable, reproducible, and scientifically valid.