Di-methyl-Histone H3.1 (K9) Recombinant Monoclonal Antibody

What is the biological significance of histone H3K9 di-methylation?

Histone H3 lysine 9 (H3-K9) methylation has been demonstrated to correlate strongly with transcriptional repression across multiple genomic contexts. This modification serves as a specific binding site for heterochromatin protein 1 (HP1), which plays a crucial role in heterochromatin formation and maintenance. The relationship between H3-K9 methylation and transcriptional repression involves multiple mechanisms, including the recruitment of repressive complexes and changes in chromatin accessibility .

Unlike some other histone modifications, H3-K9 methylation can suppress transcription through both HP1-dependent and HP1-independent mechanisms. Research has shown that H3-K9 methylation alone can inhibit transcription through a mechanism involving histone deacetylation, even in the absence of HP1 recruitment. This occurs partly because H3-K9 methylation inhibits histone acetylation by p300 without affecting its association with chromatin, illustrating the complex interplay between different histone modifications .

It's important to note that while H3-K9 methylation and HP1 are predominantly enriched in heterochromatin regions, methyl-K9 is also found in euchromatic regions that lack HP1, suggesting multiple context-dependent functions for this modification beyond heterochromatin formation .

How do different methylation states of H3K9 affect chromatin structure and gene expression?

Histone H3 can be mono-, di-, or tri-methylated by specific histone methyltransferases, with each methylation state potentially having distinct effects on gene expression. The degree of methylation at specific lysine residues creates a complex "histone code" that influences chromatin structure and accessibility. Methylation events that weaken the binding between histone tails and DNA generally lead to increased transcription by making DNA more accessible to transcription factor proteins and RNA polymerase .

In contrast, di-methylation of H3K9 is generally associated with transcriptional repression. This occurs through multiple mechanisms, including the recruitment of repressive protein complexes and the creation of condensed chromatin structures that limit accessibility to transcriptional machinery. The specific methyltransferases involved, such as SUV39H1 and G9a, can have different downstream effects despite catalyzing the same chemical modification .

Research has shown that H3-K9 methylation inhibits histone acetylation by p300, which helps explain how this modification leads to histone deacetylation in both H3 and H4. This represents a mechanistic explanation for the observed inverse relationship between histone methylation at certain residues and histone acetylation levels .

What detection methods are available for H3K9 methylation research?

Researchers have multiple methods at their disposal for detecting H3K9 methylation, each with specific advantages for different experimental questions. Western blotting represents a standard approach for quantitative analysis of histone modifications, allowing researchers to detect specific methylation states using antibodies like anti-Di-methyl-Histone H3.1 (K9). This method can be optimized to detect subtle changes in methylation levels across different experimental conditions .

Immunohistochemistry and immunofluorescence provide spatial information about H3K9 methylation patterns within individual cells. These approaches have revealed interesting localization patterns—for example, research has shown that histone H3 acetylation is often more localized to the periphery of the nucleus and diffuse within the nuclear body, while histone H4 acetylation tends to be more concentrated within the nucleus .

Flow cytometry offers another quantitative approach, allowing researchers to analyze histone modifications in large cell populations. This method can detect subtle changes in methylation levels and is particularly useful for monitoring changes over time or in response to treatments. Studies have shown that histone modifications can be reliably detected by flow cytometry over extended periods (up to 96 hours) under various temperature conditions .

What controls should be included when working with Di-methyl-Histone H3.1 (K9) antibodies?

When designing experiments using Di-methyl-Histone H3.1 (K9) antibodies, appropriate controls are essential for accurate interpretation of results. Positive controls should include samples known to contain the di-methylated H3K9 mark, such as cell lines treated with specific histone methyltransferase enhancers. For instance, the leukemia cell line 697 treated with 100 nM LBH589 has been used as a positive control in histone modification studies .

Negative controls should include samples where the modification of interest is absent or reduced. This can be achieved through treatment with histone demethylase activators or through genetic approaches that knock down specific methyltransferases. Including antibodies against total histone H3 is crucial to normalize for variations in histone content between samples .

Specificity controls are particularly important when working with histone modification antibodies. Researchers should verify that the Di-methyl-Histone H3.1 (K9) antibody doesn't cross-react with other methylation states (mono- or tri-methylation) or with the same modification at different histone residues. This can be tested using peptide competition assays with synthesized peptides containing specific modifications .

Loading controls such as β-actin (42 kDa) are essential for western blot applications to adjust for concentration variations between samples. For immunofluorescence applications, nuclear counterstains help confirm proper focusing and nuclear localization of signals .

How can I optimize ChIP protocols specifically for Di-methyl-Histone H3.1 (K9) antibodies?

Chromatin immunoprecipitation (ChIP) using Di-methyl-Histone H3.1 (K9) antibodies requires careful optimization to achieve high specificity and sensitivity. The antibody concentration is a critical parameter—for Di-methyl-Histone H3.1 (K9) monoclonal antibodies, initial dilution ratios between 1/30 to 1/200 are recommended for immunoprecipitation applications, though this should be empirically optimized for each experimental system .

Crosslinking conditions significantly impact ChIP efficiency for histone modifications. While standard protocols often use 1% formaldehyde for 10 minutes, H3K9 methylation detection may benefit from gentler crosslinking (0.5% formaldehyde for 5-8 minutes) to preserve epitope accessibility. The sonication step should be carefully optimized to generate chromatin fragments of 200-500 bp without damaging the epitopes recognized by the antibody .

Buffer composition can dramatically affect antibody performance in ChIP applications. For Di-methyl-Histone H3.1 (K9) antibodies, PBS-based buffers with pH 7.4 containing 150 mM NaCl are commonly used. The addition of 0.1% Triton X-100 or 0.05% Tween-20 can reduce non-specific binding without compromising specific antibody-epitope interactions .

Pre-clearing the chromatin with protein A/G beads before adding the antibody can significantly reduce background. Additionally, incorporating a blocking step using BSA (0.5-1%) and/or sheared salmon sperm DNA can further minimize non-specific interactions. Sequential ChIP (re-ChIP) approaches can be particularly valuable when studying the co-occurrence of H3K9 methylation with other modifications or protein interactions .

What are the methodological considerations for quantifying histone H3K9 methylation in clinical samples?

Analyzing histone H3K9 methylation in clinical samples presents unique challenges that require careful methodological consideration. Sample collection and processing significantly impact the preservation of histone modifications. Based on experimental evidence, blood samples should be processed promptly, as histone modifications can change during storage. Studies have shown that while histone acetylation remains relatively stable for up to 96 hours at 4°C and 20°C, there is a significant drop in both histone H3 and H4 acetylation after 96 hours at 37°C .

For blood samples specifically, different separation methods yield varying results. Comparisons between Ficoll-Paque separation and red cell lysis methods have demonstrated that both techniques can be effective for isolating leukocytes while preserving histone modifications, but the choice should be consistent throughout a study to avoid introducing methodological variables .

When analyzing clinical samples, normalization becomes particularly important due to potential variations in cell composition between patients. Using total histone H3 levels as a denominator provides a more accurate assessment of relative methylation levels. Additionally, including healthy control samples processed in parallel with patient samples helps control for technical variations .

Quantitative methods such as western blotting and flow cytometry are particularly valuable for clinical samples. For western blotting, membranes should be scanned and adjusted for concentration variations by normalization to reference proteins such as β-actin. Flow cytometry offers the advantage of analyzing specific cell populations within heterogeneous clinical samples, allowing for more detailed assessment of cell type-specific responses .

How do different enzymatic modulators affect H3K9 methylation patterns?

Histone H3K9 methylation is dynamically regulated by the opposing activities of histone methyltransferases (HMTs) and histone demethylases (HDMs). Two well-characterized H3-K9-specific histone methyltransferases, SUV39H1 and G9a, have distinct effects despite catalyzing the same chemical modification. Research has shown that while both enzymes induce H3-K9 methylation and repress transcription, only SUV39H1 effectively recruits heterochromatin protein 1 (HP1) to chromatin. This differential effect depends not only on the methylation activity but also on direct protein-protein interactions between SUV39H1 and HP1 .

The regulation of H3K9 methylation can be studied using various enzymatic modulators and detection methods. HTRF-based assays provide a powerful approach for screening compounds that affect HMT or HDM activity. These assays typically use a biotinylated histone H3 peptide substrate, an Eu-cryptate labeled detection antibody (such as anti-H3K9Me1), and XL665-conjugated Streptavidin. The HTRF signal is proportional to the concentration of methyl-specific peptide, allowing for quantitative assessment of enzymatic activity .

Different methylation states (mono-, di-, or tri-methylation) are regulated by specific enzymes and have distinct biological functions. The transition between these states is controlled by the balanced activities of various HMTs and HDMs. For example, LSD1 (KDM1A) specifically demethylates mono- and di-methylated H3K9, while JMJD2 family enzymes can demethylate tri-methylated H3K9. Understanding these enzymatic specificities is crucial for interpreting the biological significance of observed methylation patterns .

What are the latest approaches for simultaneous detection of multiple histone modifications?

Contemporary epigenetic research increasingly focuses on the combinatorial nature of histone modifications, necessitating methods that can detect multiple modifications simultaneously. Multiplexed immunofluorescence techniques allow researchers to visualize different histone modifications within the same cell or tissue section. These approaches have revealed important insights about the nuclear localization patterns of different modifications. For example, studies have shown that histone H3 acetylation is often more localized to the periphery of the nucleus and diffuse within the nuclear body, while other modifications show distinct distribution patterns .

Mass spectrometry-based approaches represent the state-of-the-art for comprehensive histone modification analysis. These methods can identify and quantify dozens of histone modifications simultaneously without relying on antibodies, thereby avoiding potential specificity issues. Targeted mass spectrometry approaches can be particularly valuable for analyzing specific modifications like H3K9 methylation in complex samples .

Sequential ChIP (re-ChIP) techniques involve performing successive immunoprecipitations with different antibodies to identify genomic regions that simultaneously contain multiple modifications. This approach is particularly valuable for studying the relationship between H3K9 methylation and other modifications, such as the often-inverse relationship with acetylation marks .

HTRF-based biochemical assays offer another approach for studying the interplay between different histone modifications. These assays can be designed to detect specific modification patterns using combinations of labeled antibodies. The table below illustrates the range of methylation-specific antibodies available for different histone H3 lysine residues and methylation states :

Why might I see inconsistent results when detecting Di-methyl-Histone H3.1 (K9)?

Inconsistent results when detecting Di-methyl-Histone H3.1 (K9) can stem from multiple methodological variables. Antibody specificity represents a primary concern—Di-methyl-Histone H3.1 (K9) antibodies may exhibit varying degrees of cross-reactivity with mono- or tri-methylated states or with the same modification at different lysine residues. This is particularly relevant for polyclonal antibodies, though even monoclonal antibodies may display some cross-reactivity. Researchers should verify antibody specificity using peptide competition assays with synthesized peptides containing specific modifications .

Sample preparation variables significantly impact histone modification detection. Research has demonstrated that histone modifications can change during storage, with studies showing varying stability at different temperatures. While histone acetylation remains relatively stable for up to 96 hours at 4°C and 20°C, significant changes occur after extended periods at higher temperatures. Similar principles likely apply to methylation marks, suggesting that consistent sample handling is crucial .

Technical variables in detection methods can also contribute to inconsistency. For Western blotting applications, transfer efficiency, blocking conditions, and detection reagents all impact results. Flow cytometry results can be affected by fixation conditions, permeabilization methods, and instrument settings. Standardizing these parameters and including appropriate controls in each experiment helps minimize variability .

How do sample preparation conditions affect histone methylation detection?

Sample preparation conditions critically influence the accuracy and reproducibility of histone methylation detection. Storage temperature and duration have been shown to affect histone modifications in extracted samples. Research examining histone acetylation found that samples maintained relatively stable modification levels for up to 96 hours at 4°C and 20°C, but showed significant decreases after 96 hours at 37°C. These findings suggest that histone methylation analyses should ideally be performed on fresh samples or those stored under appropriate conditions to prevent modification changes .

Extraction methods significantly impact the preservation of histone modifications. When analyzing blood samples, both Ficoll-Paque separation and red cell lysis methods can effectively isolate leukocytes while preserving histone modifications, but consistency in methodology is essential for comparative studies. The buffer composition used for cell lysis also affects epitope preservation—buffers containing protease and phosphatase inhibitors help maintain histone modifications during extraction .

Fixation conditions are particularly important for immunofluorescence and flow cytometry applications. Paraformaldehyde fixation (typically 2-4%) preserves histone modifications while maintaining cellular morphology, but overfixation can mask epitopes and reduce antibody binding. For methylation-specific antibodies like Di-methyl-Histone H3.1 (K9), brief fixation (10-15 minutes) often provides optimal results .

Sample storage buffer composition affects antibody performance in various applications. Di-methyl-Histone H3.1 (K9) antibodies are typically stored in PBS (pH 7.4) containing 150 mM NaCl, 0.02% sodium azide, and 50% glycerol. Deviations from these conditions during sample preparation or antibody dilution can affect binding specificity and signal intensity. For long-term storage, aliquoting antibodies and storing at -20°C helps prevent freeze/thaw cycles that can degrade antibody performance .

What are the common cross-reactivity issues with histone methylation antibodies?

Cross-reactivity represents a significant challenge when working with histone methylation antibodies, potentially leading to misinterpretation of experimental results. Many antibodies developed against specific histone methylation marks show some degree of cross-reactivity with related modifications. For Di-methyl-Histone H3.1 (K9) antibodies, potential cross-reactivity with mono- or tri-methylated H3K9 is a common concern. Additionally, these antibodies may recognize the same modification (di-methylation) at different lysine residues (K4, K27, or K36) due to sequence similarities in the surrounding amino acids .

The sensitivity and specificity of histone methylation antibodies can vary significantly between suppliers and even between lots from the same supplier. For critical experiments, validation using peptide competition assays with synthesized peptides containing specific modifications is highly recommended. These assays can quantitatively assess cross-reactivity with different methylation states and sites .

Cross-reactivity with non-histone proteins containing similar methylated lysine motifs is another potential issue, particularly in whole-cell lysates or complex samples. This type of cross-reactivity can be evaluated by comparing results from multiple detection methods (e.g., Western blotting, immunofluorescence, and mass spectrometry) and by including appropriate controls such as methyltransferase inhibition or genetic knockdown of specific methyltransferases .

The HTRF toolbox approach offers a method to systematically evaluate antibody specificity across multiple histone modifications. The table below illustrates the specificity profiles of various methylation-specific antibodies, highlighting the importance of selecting the appropriate antibody for each experimental question :

How can I optimize antibody dilutions for different applications?

For immunofluorescence and immunocytochemistry applications, higher antibody concentrations are generally required, with recommended dilutions between 1/30 and 1/200. This reflects the different detection sensitivity and the importance of achieving sufficient binding for visualization. When establishing optimal conditions, a dilution series spanning the recommended range should be tested on known positive control samples .

The buffer composition used for antibody dilution significantly impacts performance. For Di-methyl-Histone H3.1 (K9) antibodies, dilution in PBS containing 0.1-1% BSA or 1-5% normal serum from the same species as the secondary antibody helps reduce background without compromising specific binding. For applications involving multiple antibodies, careful optimization of each primary antibody independently is recommended before attempting multiplexed detection .

Titration experiments represent the most systematic approach to optimization. For Western blotting, this involves testing a range of antibody dilutions while keeping all other variables constant. A plot of signal-to-noise ratio versus antibody dilution typically shows a bell-shaped curve, with the optimal dilution occurring at or near the peak. Similar approaches can be applied to immunofluorescence and flow cytometry applications, with the additional consideration of minimizing cross-channel spillover in multiplexed experiments .

How does H3K9 methylation interact with other epigenetic modifications?

Histone H3K9 methylation functions within a complex network of interacting epigenetic modifications that collectively regulate chromatin structure and gene expression. Research has revealed a particularly strong inverse relationship between H3K9 methylation and histone acetylation. Studies have demonstrated that H3K9 methylation inhibits histone acetylation by p300 without affecting its association with chromatin. This mechanistic insight helps explain how H3K9 methylation leads to histone deacetylation not only in H3 but also in H4, illustrating the interconnected nature of different histone modifications .

The relationship between H3K9 methylation and DNA methylation represents another critical interaction. These modifications often co-occur at silenced genomic regions and can reinforce each other through feedback mechanisms. Proteins that recognize methylated H3K9, such as HP1, can recruit DNA methyltransferases to establish DNA methylation patterns. Conversely, methyl-CpG binding proteins can recruit histone methyltransferases to establish H3K9 methylation, creating a self-reinforcing repressive chromatin state .

H3K9 methylation states also interact with other histone methylation marks in complex ways. For example, H3K4 methylation (associated with active transcription) and H3K9 methylation (associated with repression) often show mutually exclusive patterns. Similarly, H3K9 methylation patterns interact with H3K27 methylation, another repressive mark, with some genomic regions showing both modifications while others display only one or the other .

The combinatorial patterns of these modifications create a complex "histone code" that influences chromatin structure and accessibility. Understanding these interactions requires sophisticated detection methods that can analyze multiple modifications simultaneously, such as mass spectrometry or multiplexed immunoassays .

What role does H3K9 methylation play in cellular differentiation and development?

H3K9 methylation serves as a critical epigenetic regulator during cellular differentiation and development by establishing and maintaining cell type-specific gene expression patterns. During early embryonic development, global changes in H3K9 methylation patterns occur as cells transition from totipotency to lineage commitment. These changes help establish developmental boundaries by silencing inappropriate gene expression programs while allowing lineage-specific genes to be expressed .

The dynamics of H3K9 methylation during differentiation involve both the establishment of new methylation patterns and the erasure of existing ones. This process is mediated by the balanced activities of histone methyltransferases (such as SUV39H1 and G9a) and demethylases. The specific distribution of different methylation states (mono-, di-, or tri-methylation) changes during development, with each state potentially having distinct regulatory functions .

H3K9 methylation-mediated heterochromatin formation plays a particularly important role in silencing repetitive elements and maintaining genomic stability during development. The recruitment of HP1 to H3K9-methylated regions contributes to the formation of compact chromatin structures that protect against inappropriate recombination and transposon activation, which is essential for normal development .

Research into the role of H3K9 methylation in development has been facilitated by the availability of specific antibodies against different methylation states and by advanced detection methods. These tools allow researchers to track changes in methylation patterns at specific genomic loci during differentiation and to correlate these changes with alterations in gene expression and cellular phenotype .

What are the latest findings on H3K9 methylation in disease contexts?

Aberrant H3K9 methylation patterns have been implicated in various disease processes, particularly cancer and neurodevelopmental disorders. In cancer, global changes in H3K9 methylation distribution often occur, with some regions showing hypermethylation while others display hypomethylation. These alterations contribute to inappropriate gene expression patterns, including the silencing of tumor suppressor genes and the activation of oncogenes. The enzymes regulating H3K9 methylation, such as SUV39H1 and G9a, have emerged as potential therapeutic targets, with inhibitors showing promise in preclinical studies .

Neurodevelopmental and neurodegenerative disorders have also been linked to dysregulation of H3K9 methylation. The precise establishment of neuronal gene expression patterns depends on appropriate H3K9 methylation, and disruptions to this process can lead to cognitive impairments. Research has found associations between variants in genes encoding H3K9 methylation regulators and conditions such as autism spectrum disorders and intellectual disability .

Inflammatory and autoimmune diseases represent another area where H3K9 methylation plays a role. Changes in H3K9 methylation patterns in immune cells can affect their activation state and cytokine production. Studies of patient samples have revealed alterations in global H3K9 methylation levels that correlate with disease severity in conditions such as rheumatoid arthritis and systemic lupus erythematosus .

Detection of disease-associated H3K9 methylation changes requires sensitive and specific methods. Antibody-based approaches using Di-methyl-Histone H3.1 (K9) monoclonal antibodies provide valuable tools for comparing methylation patterns between healthy and diseased tissues. When analyzing clinical samples, it's particularly important to ensure consistent sample processing and to include appropriate controls to account for technical variables .

How can Di-methyl-Histone H3.1 (K9) antibodies be used in drug discovery research?

Di-methyl-Histone H3.1 (K9) antibodies serve as essential tools in drug discovery programs targeting epigenetic regulators, particularly histone methyltransferases and demethylases. High-throughput screening assays using these antibodies can identify compounds that modulate H3K9 methylation levels. HTRF-based approaches are particularly well-suited for this application, as they allow for miniaturization to 384-well format and require no wash steps. These assays typically use a biotinylated histone H3 peptide substrate, an Eu-cryptate labeled detection antibody specific for Di-methyl-Histone H3.1 (K9), and XL665-conjugated Streptavidin .

Target validation studies benefit from Di-methyl-Histone H3.1 (K9) antibodies by allowing researchers to confirm that candidate compounds affect the intended epigenetic pathways. Western blotting, immunofluorescence, and flow cytometry using these antibodies can verify changes in H3K9 methylation levels in response to treatment with potential therapeutic agents. This is particularly important for establishing the mechanism of action of compounds identified through phenotypic screening approaches .

Pharmacodynamic biomarker development represents another important application. Changes in H3K9 methylation levels in accessible tissues (such as blood) can potentially serve as biomarkers for drug activity in clinical trials. Methods for detecting histone modifications in blood samples have been optimized, allowing for monitoring of H3K9 methylation changes in response to treatment. These approaches have shown that histone modifications can be reliably detected in clinical samples when appropriate processing methods are used .

Combination therapy approaches targeting multiple epigenetic pathways simultaneously are increasingly important in drug discovery. Di-methyl-Histone H3.1 (K9) antibodies help researchers understand how modulation of H3K9 methylation interacts with other epigenetic modifications or signaling pathways. This information guides the rational design of combination strategies that may achieve synergistic therapeutic effects while minimizing toxicity .