Tri-methyl-HIST1H3A (K36) Antibody

What is the biological significance of H3K36me3 in genomic regulation?

The positioning of H3K36me3 along gene bodies is particularly important, as it is generally associated with actively transcribed regions. Its presence helps recruit specific protein complexes that maintain chromatin states conducive to transcriptional elongation. Understanding H3K36me3 distribution patterns provides valuable insights into gene expression regulation within different cellular contexts and disease states .

How do polyclonal and monoclonal Tri-methyl-HIST1H3A (K36) antibodies differ in research applications?

The choice between polyclonal and monoclonal antibodies targeting H3K36me3 depends on specific research requirements. Polyclonal antibodies, such as those raised in rabbits (CSBPA010418PA36me3HU50 and ab9050), recognize multiple epitopes on the target antigen, potentially offering greater sensitivity in detecting H3K36me3 marks across various experimental conditions . These antibodies are particularly useful in applications where signal amplification is beneficial, such as in weakly expressed samples.

Monoclonal antibodies like MACO0062 (mouse-derived) and RM155 (rabbit-derived) provide superior specificity by recognizing a single epitope. The RM155 clone, for instance, demonstrates exceptional specificity for H3K36me3 with no cross-reactivity with non-modified K36, monomethylated K36 (K36me1), or dimethylated K36 (K36me2) . This precise recognition makes monoclonal antibodies preferred for applications requiring highly discriminative detection, such as ChIP-seq experiments where accurate genomic mapping is essential.

What sample preparation methods are optimal for H3K36me3 detection?

Optimal detection of H3K36me3 requires careful sample preparation tailored to the specific application. For Western blotting analyses, acid extraction of histones from cellular samples is highly recommended as demonstrated in protocols using RM155 antibody with HeLa cell extracts . This method efficiently enriches histone proteins while removing potential interfering components.

For immunohistochemistry and immunocytochemistry applications, appropriate fixation is crucial for epitope preservation. Formaldehyde fixation followed by permeabilization typically yields good results for H3K36me3 detection in tissue samples, as evidenced by successful staining in human normal brain tissue, colon tissue, and breast cancer specimens using the RM155 antibody .

For chromatin immunoprecipitation (ChIP) experiments, crosslinking conditions must be optimized to preserve the association between H3K36me3-modified histones and their associated DNA regions. The standard protocol typically includes:

• Crosslinking cells with 1% formaldehyde (10-15 minutes)

• Quenching with glycine

• Cell lysis and chromatin shearing (typically to 200-500bp fragments)

• Immunoprecipitation with 5μg of H3K36me3 antibody (as demonstrated with RM155)

• Washing and elution steps

• Reverse crosslinking and DNA purification for downstream analysis

How can researchers validate the specificity of H3K36me3 antibodies?

Validating antibody specificity is crucial for ensuring reliable experimental results. For H3K36me3 antibodies, several complementary approaches are recommended:

Peptide competition assays represent a primary validation method where pre-incubation of the antibody with the specific trimethylated K36 peptide should abolish signal detection, while incubation with unmodified or differently methylated peptides should not affect signal intensity. This approach directly tests the antibody's binding specificity.

Western blot validation using recombinant histone controls is another essential approach. The RM155 antibody demonstrates this validation method by showing specific binding to recombinant histone H3.3 and acid extracts of HeLa cells that contain H3K36me3, with no cross-reactivity to non-modified K36, K36me1, or K36me2 . This differential recognition pattern confirms the antibody's methylation-state specificity.

ChIP-qPCR using gene targets known to be enriched or depleted for H3K36me3 provides functional validation in the genomic context. As demonstrated with RM155, ChIP experiments on HeLa cells followed by real-time PCR with gene-specific primers confirm the ability to immunoprecipitate H3K36me3-associated DNA regions .

Testing across multiple cell types or tissues further validates consistency. The successful application of RM155 in diverse tissues including normal brain tissue, colon tissue, and breast cancer specimens demonstrates robust performance across different biological contexts .

What are the critical parameters for optimizing ChIP protocols with H3K36me3 antibodies?

Successful chromatin immunoprecipitation with H3K36me3 antibodies requires careful optimization of several parameters:

Antibody selection is paramount, with proven ChIP-grade antibodies like ab9050 (cited in over 975 publications) and RM155 being preferred options . These antibodies have been specifically validated for their performance in chromatin immunoprecipitation experiments.

Chromatin fragmentation must be optimized, as H3K36me3 typically associates with gene bodies rather than specific short sequences. Fragments of 200-500bp generally provide good resolution for H3K36me3 profiling. Sonication parameters should be carefully calibrated for each cell type to achieve consistent fragmentation.

Washing stringency affects specificity, with balanced washing conditions needed to remove non-specific interactions while preserving specific H3K36me3 binding. Typically, increasing salt concentrations in sequential washes (e.g., 150mM to 500mM NaCl) helps achieve this balance.

The inclusion of appropriate controls is essential:

• Input chromatin (pre-immunoprecipitation) controls for biases in chromatin preparation

• IgG negative controls assess non-specific binding

• Positive controls targeting regions known to be enriched for H3K36me3

• Negative controls examining regions typically lacking H3K36me3

How do different fixation methods affect H3K36me3 epitope accessibility?

Fixation methodology significantly impacts H3K36me3 epitope accessibility and detection sensitivity across different applications:

For immunohistochemistry applications, the duration of formaldehyde fixation critically affects epitope retrieval efficiency. Excessive fixation can mask the H3K36me3 epitope through over-crosslinking, while insufficient fixation may result in poor tissue preservation. Optimized protocols typically employ 10% neutral buffered formalin for 24-48 hours for tissue sections, followed by antigen retrieval methods such as heat-induced epitope retrieval in citrate buffer (pH 6.0).

In cell-based assays like immunocytochemistry, shorter fixation periods (10-15 minutes) with 4% paraformaldehyde often provide sufficient structure preservation while maintaining H3K36me3 epitope accessibility. This balanced approach has proven effective in detecting H3K36me3 in various cell types.

For ChIP applications, crosslinking duration requires careful optimization as it directly impacts chromatin shearing efficiency and epitope availability. Over-fixation can reduce antibody access to the H3K36me3 epitope and impair chromatin fragmentation, while under-fixation may fail to preserve protein-DNA interactions. A typical starting protocol uses 1% formaldehyde for 10 minutes at room temperature, with optimization based on specific cell types and experimental goals.

How can H3K36me3 antibodies be utilized in cancer research?

H3K36me3 antibodies have become instrumental in cancer research, offering insights into epigenetic mechanisms underlying oncogenesis and potential biomarker development:

In breast cancer research, immunohistochemical staining using antibodies like RM155 has revealed distinctive H3K36me3 distribution patterns that differ between normal and malignant tissues . These patterns potentially correlate with gene expression changes driving tumorigenesis and could serve as prognostic indicators.

For studying mutations in histone modifying enzymes, H3K36me3 antibodies provide a direct readout of functional consequences. In cancers harboring mutations in SETD2 (the primary methyltransferase for H3K36me3), antibody-based detection methods can quantify resulting H3K36me3 reduction and map affected genomic regions through ChIP-seq approaches.

When investigating chromatin accessibility alterations in cancer, H3K36me3 detection offers valuable contextual information. As H3K36me3 typically marks gene bodies of actively transcribed genes, changes in its distribution can indicate broader transcriptional dysregulation in cancer cells. Combining H3K36me3 ChIP-seq with RNA-seq provides powerful correlative analysis of epigenetic and transcriptomic changes.

For monitoring therapy response, tracking H3K36me3 levels using specific antibodies can assess the efficacy of epigenetic-targeting drugs. Changes in global H3K36me3 patterns may serve as early indicators of treatment response before phenotypic changes become apparent.

What are the recommended controls for validating H3K36me3 antibody specificity in different experimental contexts?

Comprehensive validation of H3K36me3 antibody specificity requires multiple control strategies across different experimental platforms:

For Western blotting applications, peptide competition assays serve as critical controls. Pre-incubation of the antibody with increasing concentrations of H3K36me3 peptide should progressively diminish signal intensity, while incubation with unmodified, mono-methylated, or di-methylated peptides should not affect signal detection. This directly demonstrates binding specificity for the trimethylated form.

In ChIP experiments, parallel immunoprecipitations using IgG matched to the H3K36me3 antibody host species establish background binding levels. Additionally, targeting regions known to lack H3K36me3 enrichment (such as inactive genes or intergenic regions) provides negative genomic controls, while actively transcribed gene bodies typically serve as positive controls for H3K36me3 enrichment.

For immunocytochemistry and immunohistochemistry, peptide blocking controls validate signal specificity. Additionally, staining tissues or cells where H3K36me3 has been experimentally reduced (through SETD2 inhibition or knockdown) provides functional validation of antibody specificity.

Knockout/knockdown controls offer the most stringent validation approach. Cells with SETD2 knockdown or knockout should display significantly reduced H3K36me3 signals across all detection platforms, confirming that the antibody is genuinely detecting this specific modification rather than cross-reacting with other epitopes.

How do H3K36me3 patterns change during cellular differentiation and in disease states?

H3K36me3 distribution undergoes significant remodeling during cellular differentiation and in various disease contexts:

During cellular differentiation, H3K36me3 patterns shift to reflect changing transcriptional programs. As certain genes become activated or repressed during differentiation, corresponding changes in H3K36me3 occupancy across these genes can be detected using techniques like ChIP-seq with specific antibodies. These dynamic changes help establish and maintain cell-type-specific gene expression patterns.

In neurodegenerative disorders, disruptions in H3K36me3 patterns have been observed. Immunohistochemical analyses of brain tissues using antibodies like RM155 can reveal altered H3K36me3 distribution that may contribute to transcriptional dysregulation in affected neurons .

Cancer cells frequently display global alterations in H3K36me3 distribution. These changes can be detected through immunohistochemistry of cancer tissues or ChIP-seq analyses of cancer cell lines using specific antibodies. For example, RM155 antibody has been successfully used to analyze H3K36me3 patterns in breast cancer specimens, revealing distinct differences compared to normal tissues .

In developmental disorders associated with mutations in histone modifying enzymes, H3K36me3 antibodies provide a valuable tool for assessing functional consequences. Reduced or altered H3K36me3 patterns may contribute to inappropriate gene expression during development, leading to phenotypic abnormalities.

What are common pitfalls in H3K36me3 antibody-based experiments and how can they be addressed?

Researchers commonly encounter several challenges when working with H3K36me3 antibodies that can be systematically addressed:

Non-specific binding in Western blots often manifests as multiple bands or high background. This can be mitigated by:

• Increasing blocking time and concentration (5% BSA or milk in TBST for 2 hours)

• Using more stringent washing conditions (higher salt or detergent concentrations)

• Further diluting primary antibody (starting with 1:5000 for Western blots with antibodies like MACO0062)

• Confirming specificity with peptide competition controls

Poor signal in immunohistochemistry applications may result from inadequate epitope retrieval. Optimization strategies include:

• Testing different antigen retrieval methods (heat-induced vs. enzymatic)

• Adjusting retrieval buffer composition and pH (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

• Extending retrieval time while monitoring tissue integrity

• Implementing signal amplification systems for low-abundance targets

Low enrichment in ChIP experiments can stem from multiple factors that should be systematically addressed:

• Optimizing crosslinking conditions (time, formaldehyde concentration)

• Improving chromatin fragmentation (sonication parameters)

• Increasing antibody amount (typically 5μg per reaction as used with RM155)

• Adjusting antibody incubation time and temperature

• Using more gentle washing conditions to preserve specific interactions

Batch-to-batch variability can be controlled through:

• Purchasing larger lots of validated antibodies

• Performing validation tests on each new lot

• Including internal controls in each experiment for normalization

• Considering monoclonal antibodies like RM155 or MACO0062 for greater consistency

How can researchers quantify H3K36me3 levels accurately across different experimental systems?

Accurate quantification of H3K36me3 levels requires tailored approaches depending on the experimental platform:

For Western blot quantification, normalization to total histone H3 is essential. This typically involves:

• Running parallel blots or sequential probing with total H3 antibody

• Using digital image analysis to determine H3K36me3/total H3 ratios

• Including recombinant histone standards with known quantities for absolute quantification

• Ensuring measurements are taken within the linear dynamic range of detection

In ChIP-qPCR approaches, implementing the percent input method provides reliable quantification:

• Processing an input sample (pre-immunoprecipitation chromatin) alongside ChIP samples

• Calculating enrichment as: % Input = 100 × 2^(Ct[Input] - Ct[ChIP])

• Including control regions (positive and negative for H3K36me3) for normalization

• Using standard curves from serial dilutions to ensure accurate quantification

For ChIP-seq quantification, several approaches ensure accurate assessment:

• Spike-in normalization with exogenous chromatin from a different species

• Normalization to regions unlikely to change in H3K36me3 status

• Integrated analysis of signal over defined genomic features (gene bodies, etc.)

• Consideration of sequencing depth and library complexity in comparative analyses

In immunohistochemistry quantification, digital pathology approaches enhance accuracy:

• Standardizing image acquisition settings across all samples

• Using automated scoring algorithms for consistent assessment

• Implementing H-score methods (staining intensity × percentage of positive cells)

• Including control tissues with known H3K36me3 status in each batch

What are the best strategies for multiplexing H3K36me3 detection with other histone modifications?

Multiplexing approaches for simultaneous detection of H3K36me3 with other histone modifications require careful experimental design:

For immunofluorescence applications, sequential or simultaneous staining protocols can be implemented:

• Using primary antibodies from different host species (e.g., rabbit anti-H3K36me3 with mouse anti-H3K4me3)

• Employing spectrally distinct fluorophore-conjugated secondary antibodies

• Including appropriate controls for antibody cross-reactivity

• Optimizing antibody dilutions to achieve comparable signal intensities

In sequential ChIP (re-ChIP) experiments, order of antibodies significantly impacts success:

• Starting with the antibody targeting the less abundant modification

• Including intermediate elution steps optimized to preserve remaining epitopes

• Verifying efficient recovery after the first immunoprecipitation

• Analyzing regions known to harbor both modifications as positive controls

For mass spectrometry-based approaches, sample preparation is critical:

• Employing specific enrichment of histone fractions

• Using appropriate digestion methods that preserve modified residues

• Implementing targeted approaches for quantification of specific modified peptides

• Including isotopically labeled standards for absolute quantification

When utilizing barcoded antibody approaches for spatial profiling:

• Validating each antibody individually before multiplexing

• Optimizing signal amplification to detect low-abundance modifications

• Implementing computational approaches to correct for potential signal bleed-through

• Including single-stained controls to establish accurate compensation parameters

How are H3K36me3 antibodies being utilized in single-cell epigenomic analyses?

H3K36me3 antibodies are increasingly being adapted for single-cell applications, opening new frontiers in understanding epigenetic heterogeneity:

In single-cell CUT&Tag protocols, H3K36me3 antibodies enable profiling of this modification in individual cells. This approach involves:

• Binding of the H3K36me3 antibody to intact cells or nuclei

• Addition of protein A-Tn5 transposase complex

• Targeted tagmentation of DNA adjacent to antibody binding sites

• Single-cell barcoding and sequencing

• Computational integration with transcriptomic data

For microscopy-based approaches in single cells, high-specificity antibodies like RM155 are valuable for:

• Visualizing H3K36me3 distribution within individual nuclei

• Correlating spatial patterns with cell cycle phase or differentiation status

• Quantifying cell-to-cell variation in H3K36me3 levels

• Combining with RNA FISH to directly correlate modification status with gene expression

When implementing microfluidic-based single-cell epigenomic platforms, H3K36me3 antibodies support:

• Droplet-based processing of individual cells

• Antibody-based chromatin capture

• Integration with other single-cell omics modalities

• Trajectory analyses of H3K36me3 changes during cellular transitions

For computational integration approaches, H3K36me3 profiles provide valuable layers for multi-omic analyses:

• Correlating H3K36me3 patterns with single-cell transcriptomes

• Identifying regulatory relationships at single-cell resolution

• Reconstructing epigenetic trajectories during differentiation or disease progression

• Defining cell states based on integrated epigenetic and transcriptional profiles

What are the latest developments in ChIP-seq protocols using H3K36me3 antibodies?

Recent advances in ChIP-seq protocols with H3K36me3 antibodies have enhanced sensitivity, specificity, and throughput:

Low-input ChIP-seq protocols have been developed, allowing H3K36me3 profiling from limited samples:

• Optimized chromatin preparation from as few as 1,000 cells

• Enhanced antibody capture efficiency through improved beads and buffers

• Specialized library preparation methods for low DNA amounts

• Computational approaches to handle increased technical noise

For automated ChIP platforms, H3K36me3 antibodies have been successfully implemented:

• Standardized protocols for consistent results across experiments

• Reduced technical variability through precise timing and handling

• Higher throughput allowing for larger experimental designs

• Integrated quality control metrics for reliable data generation

When implementing spike-in normalization strategies, H3K36me3 ChIP benefits from:

• Addition of foreign chromatin (e.g., Drosophila) prior to immunoprecipitation

• Use of antibodies like RM155 that recognize H3K36me3 across species

• Computational normalization based on spike-in recovery

• More accurate quantification of global changes in H3K36me3 levels

For direct combinatorial indexing approaches, H3K36me3 antibodies support:

• Parallel processing of multiple samples in a single tube

• Combinatorial barcoding strategies for high-throughput profiling

• Integration with other histone modifications for multi-dimensional analyses

• Cost-effective experimental designs for large-scale studies

How do researchers integrate H3K36me3 ChIP-seq data with other genomic datasets for comprehensive epigenetic analysis?

Integrative analysis approaches maximize the value of H3K36me3 ChIP-seq data when combined with complementary genomic datasets:

For integration with transcriptomic data, several approaches prove valuable:

• Correlation of H3K36me3 levels over gene bodies with RNA-seq expression values

• Analysis of H3K36me3 distribution patterns relative to exon-intron boundaries

• Investigation of co-transcriptional processes through integrated visualization

• Machine learning models to predict expression based on H3K36me3 patterns

When combining with chromatin accessibility data (ATAC-seq, DNase-seq):

• Contrasting H3K36me3-enriched regions with accessibility patterns

• Identifying transcription factor binding sites within H3K36me3-marked regions

• Characterizing the relationship between chromatin accessibility and transcriptional activity

• Developing integrative models of chromatin state

For multi-modification histone ChIP-seq integration:

• Implementing comprehensive chromatin state analyses (e.g., ChromHMM)

• Characterizing combinatorial patterns of histone modifications

• Identifying genomic regions with unique combinations of modifications

• Correlating modification patterns with functional genomic elements

When integrating with three-dimensional chromatin organization data:

• Examining H3K36me3 distribution within topologically associating domains (TADs)

• Correlating H3K36me3 patterns with A/B compartmentalization

• Investigating the relationship between active transcription, H3K36me3, and chromatin looping

• Developing multi-layer models of genome organization and function