Acetyl-HIST1H2BC (K12) Antibody

What is HIST1H2BC and what role does acetylation at K12 play in epigenetic regulation?

HIST1H2BC (Histone H2B type 1-C/E/F/G/I) is a core component of nucleosomes that plays a central role in chromatin structure and gene regulation. Acetylation of histone H2B at lysine 12 (K12) is a post-translational modification associated with active gene transcription and is involved in various cellular processes including DNA repair and replication . This specific modification contributes to the "histone code" that regulates DNA accessibility to transcription machinery .

Mechanistically, H2B K12 acetylation promotes chromatin relaxation by neutralizing the positive charge of the lysine residue, thereby reducing the electrostatic interaction between histones and negatively charged DNA. This modification is particularly important for gene activation and chromatin remodeling processes .

How does Acetyl-HIST1H2BC (K12) differ from other histone acetylation marks?

Acetyl-HIST1H2BC (K12) represents a specific acetylation site among multiple possible acetylation marks on histones. While H2B can be acetylated at various lysine residues (including K5, K15, K20, K24, K116, and K120), each modification site has distinct functional implications .

Unlike H4 acetylation at K5 and K12, which is primarily associated with newly synthesized histones and chromatin assembly , H2B K12 acetylation is more directly involved in active gene transcription. It differs from acetylation at H2B K120, which is more specifically implicated in nucleosome stability and DNA damage response pathways . The H2B K12 acetylation also has different genomic distribution patterns compared to other histone marks like H3K9 acetylation, which is often found at active promoters .

What are the technical specifications and validated applications for Acetyl-HIST1H2BC (K12) antibodies?

The commercially available Acetyl-HIST1H2BC (K12) antibodies have been validated for multiple research applications with specific technical parameters:

These antibodies have been particularly validated for detecting the specific acetylation at K12 of Histone H2B in human samples across various experimental setups .

What are the optimal protocols for using Acetyl-HIST1H2BC (K12) antibody in ChIP and ChIP-seq experiments?

For Chromatin Immunoprecipitation (ChIP) experiments using Acetyl-HIST1H2BC (K12) antibody, researchers should follow these optimized protocols:

Sample Preparation:

• Cross-link protein-DNA complexes using 1% formaldehyde for 10 minutes at room temperature

• Quench with 0.125M glycine for 5 minutes

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

Immunoprecipitation:

• Pre-clear chromatin with protein A/G beads

• Incubate 1-5 μg of Acetyl-HIST1H2BC (K12) antibody with chromatin overnight at 4°C

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

• Wash stringently to reduce background

• Elute and reverse cross-links

• Purify DNA for qPCR or sequencing

For ChIP-seq specifically, 0.5-1 μg of antibody is recommended for approximately 1.5 million cells to achieve optimal signal-to-noise ratio . When analyzing ChIP-seq data, researchers should note that H2B K12ac marks are typically enriched around transcription start sites of actively transcribed genes.

ChIP-qPCR validation experiments have demonstrated significant enrichment of Acetyl-HIST1H2BC (K12) at promoters of actively transcribed genes like GAPDH and ACTB compared to inactive regions like MYOD1 or Sat2 satellite repeats .

How should Western blot protocols be optimized for detecting Acetyl-HIST1H2BC (K12)?

For optimal Western blot detection of Acetyl-HIST1H2BC (K12):

Sample Preparation:

• Extract histones using an acid extraction method (e.g., 0.2N HCl) to enrich for histone proteins

• Alternatively, use whole cell lysates with HDAC inhibitors (e.g., sodium butyrate, TSA) in the lysis buffer to preserve acetylation marks

• Use 15-25 μg of histone extract or 25-50 μg of whole cell lysate

Electrophoresis and Transfer:

• Use 15-18% SDS-PAGE gels to properly resolve the low molecular weight histone proteins

• Transfer to PVDF membranes at lower voltage (30V) overnight at 4°C to ensure efficient transfer of small proteins

Antibody Incubation:

• Block with 5% non-fat milk or BSA in TBST for 1 hour

• Incubate with Acetyl-HIST1H2BC (K12) antibody at 1:100-1:1000 dilution overnight at 4°C

• Use a compatible secondary antibody (typically anti-rabbit HRP) at 1:5000-1:10000 dilution

Detection:

• Use enhanced chemiluminescence (ECL) reagents

• The expected molecular weight for Histone H2B is approximately 14 kDa

Western blot analysis has successfully detected Acetyl-HIST1H2BC (K12) in various cell lines including HeLa and 293 whole cell lysates .

What controls should be included when using Acetyl-HIST1H2BC (K12) antibody to ensure specificity?

To ensure experimental validity and antibody specificity, include these essential controls:

Positive Controls:

• Histone extracts from cells treated with HDAC inhibitors (e.g., TSA, sodium butyrate) to increase global acetylation levels

• Recombinant acetylated H2B peptides or proteins (if available)

• Cell lines known to have high levels of H2B K12 acetylation (e.g., HeLa cells)

Negative Controls:

• Samples treated with HDAC enzymes to remove acetylation marks

• Immunoprecipitation with isotype-matched IgG for ChIP experiments

• Peptide competition assays using the immunizing peptide to demonstrate specificity

Specificity Validation:

• Dot blot analysis using peptides containing unmodified H2B and other histone modifications

• Western blot against recombinant unmodified H2B to show acetylation specificity

• Immunofluorescence with peptide competition to confirm specific nuclear staining

To verify antibody specificity, researchers have performed dot blot analyses with gradually increasing concentrations (0.2-100 pmol) of modified and unmodified peptides, showing that anti-Acetyl-HIST1H2BC (K12) antibodies specifically recognize the acetylated form but not the unmodified form .

How specific are Acetyl-HIST1H2BC (K12) antibodies, and what cross-reactivities should researchers be aware of?

Understanding antibody specificity is crucial for accurate data interpretation. For Acetyl-HIST1H2BC (K12) antibodies:

Cross-reactivity Concerns:

• Pan-K-acetyl antibodies may recognize multiple acetylation sites beyond K12

• Studies have shown that some pan-K-acyl antibodies exhibit cross-reactivity between acetylation, crotonylation, and butyrylation modifications

• In dot-blot assays, while high specificity is observed at low exposure times, longer exposures may reveal cross-reactivity with other acylated lysines

In competition assays, acetyl-BSA can outcompete signals from pan-K-crotonyl and pan-K-butyryl antibodies, indicating potential cross-reactivity issues that researchers should be aware of . This is particularly important since the amount of histone acetylation exceeds other non-acetyl acylations by at least 200 times in eukaryotic cells .

To address specificity concerns, researchers should:

• Include appropriate controls (as mentioned in section 2.3)

• Consider using orthogonal methods to validate findings

• Be cautious when interpreting results from experiments using pan-K-acyl antibodies

Can Acetyl-HIST1H2BC (K12) antibodies distinguish between different H2B variants?

Histone H2B has multiple variants with high sequence homology, particularly around the K12 position. The HIST1H2BC (K12) antibody's ability to distinguish between variants depends on:

• Sequence conservation: The K12 region is highly conserved among H2B variants including HIST1H2BC, HIST1H2BE, HIST1H2BF, HIST1H2BG, and HIST1H2BI

• Antibody design: Most commercially available antibodies are raised against peptide sequences that encompass multiple H2B variants, making them unable to distinguish between these specific variants

• Target specificity: The antibodies primarily recognize the acetylation state at K12 rather than distinguishing between different H2B variants

Researchers should be aware that:

• The antibody will detect K12 acetylation across multiple H2B variants with similar sequences around K12

• For variant-specific detection, specialized antibodies targeting unique regions of specific H2B variants would be required

• Mass spectrometry approaches may be better suited for distinguishing between acetylated variants if variant specificity is crucial to the research question

How can Acetyl-HIST1H2BC (K12) antibodies be used to study the relationship between metabolism and epigenetic regulation?

Recent research has established important connections between cellular metabolism and histone acetylation, with significant implications for using Acetyl-HIST1H2BC (K12) antibodies in metabolic epigenetics studies:

Metabolic-Epigenetic Connection:

• Acetyl groups incorporated into histone modifications are predominantly produced de novo from glucose, the primary nutrient utilized by proliferating cells

• Two key metabolic enzymes control acetyl groups destined for chromatin modifications: ATP citrate lyase (ACLY) and Acyl-CoA short chain synthetase family member 2 (ACSS2)

• HAT1 (Histone Acetyltransferase 1) integrates nutrient availability and places available acetyl groups on nascent histones

Experimental Approaches:

• Metabolic manipulation experiments: Researchers can use glucose or acetate restriction/supplementation followed by Acetyl-HIST1H2BC (K12) immunoblotting to assess how metabolic changes affect H2B acetylation levels

• ChIP-seq under varying metabolic conditions: Perform ChIP-seq using Acetyl-HIST1H2BC (K12) antibodies under normal, glucose-deprived, and metabolically stressed conditions to map changes in genome-wide acetylation patterns

• Combined metabolomics and ChIP approaches: Correlate acetyl-CoA levels with H2B K12 acetylation patterns across different metabolic states

Research has shown that HAT1-induced H4 di-acetylation marks are strongly dependent on adequate glucose levels, suggesting that histone acetylation, including H2B K12ac, could serve as a glucose-sensing mechanism . Acetyl-HIST1H2BC (K12) antibodies can be instrumental in studying this nutrient-sensitive epigenetic regulation.

What is the role of H2B K12 acetylation in DNA damage response, and how can the antibody be used to study this process?

H2B K12 acetylation plays a specific role in DNA damage response (DDR) pathways:

Relationship to DNA Damage Response:

• Histone modifications, including H2B acetylation, are critical for regulating chromatin accessibility during DNA repair processes

• The DDR involves complex signaling cascades including phosphorylation of H2AX (γH2AX), followed by recruitment of MDC1, RNF8, and RNF168

• H2B acetylation may facilitate chromatin relaxation to allow access of repair machinery to damaged DNA

Experimental Approaches:

• Co-localization studies: Use immunofluorescence with Acetyl-HIST1H2BC (K12) and γH2AX antibodies to assess co-localization at DNA damage foci following treatment with DNA-damaging agents

• ChIP-seq after DNA damage: Map genome-wide changes in H2B K12 acetylation patterns after inducing DNA damage with agents like ionizing radiation, UV, or radiomimetic drugs

• Time-course experiments: Monitor dynamics of H2B K12 acetylation during the DNA damage response and repair process

• Manipulation of HATs/HDACs: Assess how inhibition or depletion of specific histone acetyltransferases or deacetylases affects H2B K12 acetylation and DNA repair efficiency

By combining these approaches, researchers can elucidate the specific role of H2B K12 acetylation in facilitating or regulating DNA repair processes.

What are common issues encountered when using Acetyl-HIST1H2BC (K12) antibodies and how can they be resolved?

Researchers frequently encounter these challenges when working with Acetyl-HIST1H2BC (K12) antibodies:

Issue 1: Weak or No Signal in Western Blots
Possible Causes and Solutions:

• Insufficient histone extraction: Use acid extraction methods (0.2N HCl) to enrich for histones

• Acetylation loss during sample preparation: Add HDAC inhibitors (10mM sodium butyrate, 1μM TSA) to all buffers

• Insufficient antibody concentration: Increase antibody concentration to 1:100-1:500

• Transfer issues: Use PVDF membranes and transfer at lower voltage (30V) overnight

• Detection sensitivity: Use high-sensitivity ECL substrates or increase exposure time

Issue 2: High Background in Immunofluorescence
Possible Causes and Solutions:

• Insufficient blocking: Increase blocking time to 2 hours and use 5% BSA

• High antibody concentration: Optimize dilution (start with 1:10 and adjust as needed)

• Secondary antibody cross-reactivity: Use highly cross-adsorbed secondary antibodies

• Fixation issues: Compare paraformaldehyde vs. methanol fixation

• Autofluorescence: Include a quenching step (e.g., 0.1% sodium borohydride)

Issue 3: Poor Enrichment in ChIP Experiments
Possible Causes and Solutions:

• Insufficient chromatin shearing: Optimize sonication conditions

• Low acetylation levels: Consider HDAC inhibitor treatment of cells

• Antibody specificity issues: Validate antibody using dot blot with acetylated peptides

• High background binding: Include more stringent wash steps

• Cross-linking efficiency: Optimize formaldehyde concentration and cross-linking time

Issue 4: Cross-Reactivity with Other Acetylation Sites
Possible Causes and Solutions:

• Antibody specificity: Perform peptide competition assays

• Dot blot validation: Test against various acetylated peptides

• Consider validation with immunodepleted samples

How can researchers validate antibody specificity when studying H2B K12 acetylation in different experimental contexts?

Comprehensive validation of Acetyl-HIST1H2BC (K12) antibodies is essential across different experimental platforms:

Western Blot Validation:

• Peptide competition assays: Pre-incubate antibody with acetylated K12 peptides

• HDAC inhibitor treatment: Compare acetylation levels in treated vs. untreated samples

• HAT depletion experiments: Examine acetylation in cells with reduced HAT activity

• Recombinant protein controls: Include unmodified and K12-acetylated recombinant H2B

ChIP Validation:

• Spike-in controls: Use exogenous chromatin with known acetylation status

• Sequential ChIP: Perform ChIP with anti-H2B followed by anti-acetyl K12

• Positive/negative control regions: Include genes known to be enriched/depleted for H2B K12ac

• Technical replicates: Demonstrate reproducibility across independent experiments

Immunofluorescence Validation:

• Peptide blocking: Pre-incubate antibody with immunizing peptide

• HDAC inhibitor treatment: Show increased signal in treated cells

• Subcellular localization: Confirm expected nuclear localization

• Colocalization with other histone marks: Verify spatial patterns match expectations

Mass Spectrometry Validation:
For ultimate validation, researchers can use targeted mass spectrometry approaches to definitively identify and quantify H2B K12 acetylation, then correlate MS findings with antibody-based detection methods.

How does Acetyl-HIST1H2BC (K12) compare with other acetylation sites on H2B in terms of biological significance and detection methods?

H2B can be acetylated at multiple lysine residues, each with distinct functional implications:

Research has shown that K12 and K5 acetylation often co-occur and have related functions, while acetylation at K116 and K120 may have distinct roles in chromatin remodeling during transcription and DNA repair .

When designing experiments, researchers should consider:

• The genomic distribution of different acetylation marks varies, with K12ac more enriched at active promoters

• Antibody specificity issues can be more pronounced for some sites than others

• Combined analysis of multiple acetylation sites provides more comprehensive insights into chromatin regulation

What emerging techniques are enhancing the study of H2B K12 acetylation beyond traditional antibody-based approaches?

Beyond traditional antibody-based detection, innovative approaches are advancing H2B K12 acetylation research:

CUT&RUN and CUT&Tag:
These techniques offer advantages over traditional ChIP by providing higher resolution, requiring fewer cells, and reducing background. They can be adapted for H2B K12ac detection using the same antibodies but with improved sensitivity and specificity.

Single-Cell Epigenomics:
Recent advances allow examination of H2B K12 acetylation at single-cell resolution, revealing cell-to-cell variability in acetylation patterns that may correlate with transcriptional heterogeneity.

Live-Cell Imaging of Acetylation:
Genetically encoded acetylation sensors and FRET-based approaches can monitor real-time dynamics of histone acetylation, including potentially H2B K12ac.

Metabolic Labeling and Click Chemistry:
These techniques allow tracking newly deposited acetylation marks by using modified acetyl-CoA precursors that can be labeled and tracked, enabling temporal studies of H2B K12 acetylation dynamics.

CRISPR-Based Epigenome Editing:
Site-specific manipulation of H2B K12 acetylation using CRISPR-dCas9 fused to histone acetyltransferases allows researchers to directly test the functional consequences of this modification at specific genomic loci.

These emerging technologies complement traditional antibody-based approaches and offer new insights into the dynamics and functional significance of H2B K12 acetylation in various biological contexts.