Histone H3K9me2 Antibody

What is H3K9me2 and why is it significant in epigenetic research?

H3K9me2 is a posttranslational histone modification that specifically marks heterochromatin at the nuclear periphery. Immunostaining studies show that while H3K9me3 and H3K27me3 co-localize with heterochromatin in the nuclear interior or at both interior and periphery, H3K9me2 distinctively marks only peripheral heterochromatin . This modification plays a critical role in organizing nuclear architecture, with close association between H3K9me2 and the nuclear lamina marker Lamin B observed in both single-cell immunostaining and ChIP-seq data correlation studies .

The modification is regulated by different histone methyltransferases (HMTs) in different nuclear compartments. In embryonic stem cells (ESCs) and differentiated mammalian cells, A-compartment H3K9me2 levels depend on SETDB1 and particularly on G9A/GLP, while B-compartment levels depend on all five main HMTs (G9A, GLP, SETDB1, SUV39H1, and SUV39H2) . Deregulation of H3K9me2 can lead to impaired cell differentiation, loss of tissue identity, premature aging, and cancer development .

Recent clinical research has identified H3K9me2 as a key biomarker in cancer biology. A study investigating breast cancer subtypes found H3K9me2 to be an independent predictor for distinguishing triple-negative breast cancer (TNBC) from less aggressive breast cancer subtypes, with elevated expression correlating with higher tumor grade and stage .

How do researchers validate the specificity of H3K9me2 antibodies?

Comprehensive validation of H3K9me2 antibodies is essential due to the challenge of distinguishing between closely related histone modifications. A robust validation approach includes:

• Peptide competition assays: Preincubate the antibody with peptides representing various histone modifications before immunostaining. As demonstrated in Figure 2 from Poleshko et al., the H3K9me2 antibody should only be blocked by the H3K9me2 peptide and not by other modifications .

• Signal intensity analysis: Compare specific peripheral nuclear staining (H3K9me2-specific signal) with nonspecific background signal in the nuclear interior using line intensity profiles .

• Super-resolution microscopy: Use techniques like STORM (Stochastic Optical Reconstruction Microscopy) imaging with peptide blocking to further validate specificity .

• Peptide arrays: Test antibody binding against a comprehensive panel of histone peptides representing various modifications. The Histone Antibody Specificity Database provides data on commercial antibody performance on peptide microarrays .

• Western blot validation: Verify that the antibody detects a single band corresponding to histone H3 molecular weight .

• Multiple antibody comparison: Compare results using antibodies from different vendors or clones. For example, studies have compared data from antibodies like Active Motif #39239 and Abcam #ab1220 .

What are the typical applications for H3K9me2 antibodies in epigenetic research?

H3K9me2 antibodies are versatile tools in epigenetic research with applications across multiple techniques:

• Chromatin Immunoprecipitation (ChIP): H3K9me2 antibodies are extensively used in ChIP assays to identify genomic regions associated with this modification. Both standard ChIP and ChIP-seq applications have been validated for multiple commercial antibodies .

• Immunofluorescence/Immunocytochemistry: These antibodies enable visualization of H3K9me2 distribution patterns in the nucleus. The characteristic peripheral nuclear staining pattern serves as a validation point for antibody specificity .

• Western Blotting: For quantifying global levels of H3K9me2 in cell or tissue lysates, providing a single band at approximately 17 kDa corresponding to histone H3 .

• Live-cell imaging: Using specialized approaches like Fab-based live endogenous modification labeling (FabLEM), researchers can track H3K9me2 dynamics in living cells without disturbing cell growth or embryo development .

• Immunohistochemistry: For examining H3K9me2 patterns in tissue sections, which has proven valuable in clinical research contexts such as cancer biomarker studies .

• Dot Blot Assays: To test antibody specificity against purified histones or histone peptides bearing specific modifications .

• FRAP Analysis: Fluorescence Recovery After Photobleaching studies using labeled antibody fragments help determine binding kinetics to H3K9me2 in living cells .

How can researchers address cross-reactivity issues with H3K9me2 antibodies?

Cross-reactivity is a significant concern with histone modification antibodies, as histone tails contain many similar sequence motifs with different modifications. Advanced approaches to identify and mitigate cross-reactivity include:

Identification methods:

• Comprehensive peptide blocking experiments: Testing specificity against an array of modified peptides. Studies show that preincubating anti-H3K9me2 antibody with peptides representing each possible histone tail modification can determine that it detects only the dimethyl modification on lysine 9 of histone H3 .

• Signal intensity spectral analysis: As demonstrated in Figure 2B of search result , this approach can distinguish specific H3K9me2 signal (at nuclear periphery) from non-specific background signal (in nuclear interior).

• Epitope analysis: Investigating whether the antibody binding is affected by adjacent modifications. For example, testing if H3K9me2 antibody binding is blocked by H3K9me2S10p peptide would reveal sensitivity to neighboring modifications .

• Off-target PTM investigation: The Histone Antibody Specificity Database identified some H3K9me3 antibodies that cross-react with other tri-methylated lysine residues, including H3K27me3, H3K23me3, and H3K18me3 . Similar assessments should be performed for H3K9me2 antibodies.

Mitigation strategies:

• Use multiple independent antibodies: Compare results from different antibody clones to identify consistent signals.

• Include appropriate negative controls: Such as IgG controls in ChIP experiments or peptide-blocked antibody controls in immunostaining.

• Validation in knockout/knockdown systems: Using cells with reduced levels of the relevant histone methyltransferases (G9A/GLP for H3K9me2) can provide definitive validation.

• Combined approaches: Integrate antibody-based detection with orthogonal techniques like mass spectrometry.

What are the optimal protocols for H3K9me2 ChIP-seq experiments?

ChIP-seq with H3K9me2 antibodies requires careful optimization. The following protocol elements are critical:

Chromatin preparation:

• Fixation: 1% formaldehyde for 5-10 minutes at room temperature is optimal, as demonstrated in multiple studies .

• Quenching: 125 mM glycine effectively stops fixation.

• Sonication: Optimize to achieve fragments of 200-500 bp for optimal immunoprecipitation and sequencing.

Immunoprecipitation:

• Antibody titration: Generate a chromatin-antibody binding isotherm to determine optimal concentration. Research shows H3K9me2 IP typically reaches saturation at around 10 μg of antibody .

• Chromatin input: 5-25 μg of chromatin is typically used per IP reaction.

• Controls: Include input control, IgG control, and consider spike-in controls for quantitative comparisons.

Data analysis considerations:

• Normalization: Quantitative ChIP-seq data can be generated without spike-in normalization by using appropriate antibody concentrations in the saturating range .

• Domain calling: H3K9me2 often forms broad domains rather than sharp peaks, requiring appropriate computational approaches.

• Integration: Compare with other heterochromatin marks and nuclear lamina association data.

Antibody performance considerations:

• Different commercial H3K9me2 antibodies show variation in ChIP efficiency. For example, Abcam ab1220 (mAbcam 1220) has been cited in over 960 publications and extensively validated for ChIP applications .

• Native ChIP vs. crosslinked ChIP: Some H3K9me2 antibodies perform differently under these conditions, so pilot experiments comparing both approaches may be valuable .

How can researchers accurately quantify global H3K9me2 levels?

Accurate quantification of global H3K9me2 levels presents several challenges. Advanced methodological approaches include:

Western blot quantification:

• Use acid-extracted histones to enrich for histone proteins.

• Include loading controls (e.g., total H3) for normalization.

• Apply a standard curve using recombinant histones for absolute quantification.

• Compare signals across multiple commercial antibodies to ensure consistency.

Flow cytometry:

• Offers single-cell resolution of global H3K9me2 levels.

• Requires extensive validation of fixation and permeabilization conditions.

• Include appropriate isotype controls and blocking steps.

Live-cell imaging approaches:

• The FabLEM (Fab-based live endogenous modification labeling) method uses fluorescently labeled specific antigen binding fragments (Fabs) to monitor H3K9me2 levels in living cells .

• The ratio of bound and free molecules depends on target concentration, allowing measurement of changes in global modification levels .

• FRAP analysis reveals binding dynamics, with FabH3K9me2 showing characteristic recovery times that reflect the abundance and accessibility of the modification .

Mass spectrometry:

• Provides absolute quantification without antibody bias.

• Can be integrated with antibody-based approaches for validation.

ChIP-seq quantification:

• Antibody titration experiments enable quantitative assessment of chromatin-antibody binding isotherms .

• The reproducibility of binding isotherms provides a landmark for consistent experimental design .

How does H3K9me2 distribution correlate with nuclear architecture and gene expression?

H3K9me2 shows distinct nuclear localization patterns that correlate with genome organization and gene regulation:

Nuclear localization:

• Immunostaining demonstrates that H3K9me2 specifically marks peripheral heterochromatin, in contrast to H3K9me3 and H3K27me3 which mark interior heterochromatin or both compartments .

• H3K9me2 closely associates with the nuclear lamina marker Lamin B, consistent with its enrichment at Lamina-Associated Domains (LADs) .

Genome compartmentalization:

• H3K9me2 is enriched at LADs, which significantly overlap with the B compartment in genome organization studies .

• Different histone methyltransferases regulate H3K9me2 in different nuclear compartments: A-compartment H3K9me2 levels depend primarily on G9A/GLP, while B-compartment levels depend on all five main HMTs .

Gene expression correlation:

• H3K9me2 is generally associated with gene repression and maintenance of heterochromatin.

• In breast cancer studies, elevated H3K9me2 levels correlate with higher tumor grade and stage in triple-negative breast cancer, suggesting a role in regulating genes involved in cancer progression .

• G9a inhibition (which reduces H3K9me2 levels) has been shown to impair cell proliferation and modulate epithelial-mesenchymal transition pathways in cancer models .

Dynamics during cellular differentiation:

• The separation of A and B compartments is less pronounced in undifferentiated embryonic stem cells (ESCs), and changes in H3K9 methylation patterns occur during differentiation .

• SETDB1 ablation in ESCs results in changes in H3K9 methylation in both A and B compartments .

How do different commercial H3K9me2 antibodies compare in performance across applications?

Several commercial H3K9me2 antibodies are widely used, with varying performance characteristics:

Abcam ab1220 (mAbcam 1220):

• Mouse monoclonal antibody

• Extensively validated for ChIP, WB, IF applications

• Cited in over 960 publications since 2005

• Demonstrates specific peripheral nuclear staining pattern in immunofluorescence

• Western blot shows a single band at approximately 17 kDa

• Validated against peptide arrays to confirm specificity

Abcam ab176882 (EP16990):

• Rabbit recombinant monoclonal antibody

• Suitable for ChIP, WB, peptide arrays, ICC/IF, IHC-P

• Recombinant production may offer improved lot-to-lot consistency

• Validated for human, mouse, and rat samples

Active Motif #39753:

• Purified IgG raised against a peptide including dimethyl-lysine 9 of histone H3

• Validated for ChIP, WB, IF applications

• Recommended for use with ChIP-IT High Sensitivity Kit or magnetic bead-based ChIP-IT Express Kits

Abbexa polyclonal antibody:

• Rabbit polyclonal antibody

• Reactive to human, mouse, and rat samples

• Validated for ELISA, WB, IHC, IF/ICC, Dot Blot, and ChIP

• Recommended dilutions: 1/500-1/1000 for WB, 1/50-1/200 for IHC-P and IF/ICC

Performance comparison table:

What methodological approaches can detect changes in H3K9me2 during cellular differentiation or disease progression?

Tracking H3K9me2 changes during biological processes requires sophisticated methodological approaches:

Time-course ChIP-seq:

• Sequential sampling during differentiation or disease progression

• Requires careful normalization strategies to compare across time points

• Can reveal dynamic changes in H3K9me2 distribution patterns

• Integration with transcriptome data can correlate epigenetic changes with gene expression

Live-cell imaging:

• FabLEM approach allows monitoring of H3K9me2 in living cells without disturbing cell growth or embryo development

• High-affinity Fabs are suitable for mouse embryo imaging, enabling monitoring of histone modifications in preimplantation embryos

• FRAP analysis provides insights into binding dynamics and global modification levels

Single-cell approaches:

• Single-cell ChIP technologies can reveal cell-to-cell variation in H3K9me2 patterns

• Immunofluorescence combined with high-content imaging allows quantification at single-cell resolution

• Flow cytometry with H3K9me2 antibodies enables high-throughput single-cell analysis

Clinical tissue analysis:

• Immunohistochemistry with validated H3K9me2 antibodies can assess modification levels in patient samples

• H3K9me2 has shown value as a biomarker in cancer studies, particularly in distinguishing triple-negative breast cancer from less aggressive subtypes

• Quantitative image analysis of IHC staining enables correlation with clinical parameters

Drug response monitoring:

• H3K9me2 levels can be monitored in response to epigenetic therapies

• For example, G9a inhibition has demonstrated anti-cancer effects by modulating H3K9me2 levels and disrupting oncogenic pathways, particularly in triple-negative breast cancer models