Tri-methyl-Histone H3(K36) Monoclonal Antibody
Description
Definition and Biological Context
Histone H3 is a core component of nucleosomes, which package DNA into chromatin. The tri-methylation of lysine 36 on histone H3 (H3K36me3) is associated with transcriptional elongation, suppression of cryptic transcription, and DNA damage repair . Monoclonal antibodies targeting H3K36me3 enable precise detection of this modification across experimental workflows.
Antibody Development and Specificity
Tri-methyl-Histone H3(K36) monoclonal antibodies are generated using synthetic peptides or recombinant proteins as immunogens. Key clones include:
These antibodies exhibit high affinity and are validated for minimal cross-reactivity with other histone modifications .
Chromatin Immunoprecipitation (ChIP)
-
RM155 (RevMab) has been used in ChIP assays to study H3K36me3 enrichment at gene bodies, revealing its role in suppressing intragenic transcription initiation .
-
In fission yeast, H3K36me3 antibodies helped demonstrate reduced methylation in H3-G34R/V mutants, linking histone mutations to transcriptional dysregulation .
Western Blotting (WB)
-
Antibodies like MACO0062 detect H3K36me3 in acid-extracted histones from HeLa cells, with recommended dilutions of 1:500–1:5,000 .
Immunofluorescence (IF) and Immunohistochemistry (IHC)
-
RM155 shows strong nuclear staining in normal human brain, colon, and breast cancer tissues, highlighting cell-type-specific H3K36me3 distribution .
Role in Cancer Biology
-
H3K36me3 loss in H3-G34R/V mutants correlates with defective DNA repair and chromosomal instability, contributing to oncogenesis .
-
PRDM9, a methyltransferase, trimethylates H3K36 and H3K4, implicating it in recombination hotspots and cancer susceptibility .
Epigenetic Regulation
-
Set2-mediated H3K36me3 in fission yeast ensures proper nucleosome remodeling and transcriptional fidelity .
-
Mass spectrometry confirmed that H3-G34R mutants reduce H3K36ac and H3K36me3, whereas G34V mutants only impair H3K36me3 .
Validation
Product Specs
Q&A
What experimental applications are tri-methyl-H3K36 monoclonal antibodies validated for, and how should researchers optimize their use?
Tri-methyl-H3K36 monoclonal antibodies (e.g., MACO0062, clone MC86) are primarily validated for:
-
Western blotting: Optimal dilution ranges between 1:500–1:5,000, depending on sample type and abundance of H3K36me3 .
-
Immunofluorescence: Requires antigen retrieval with citrate buffer (pH 6.0) and fixation with 4% paraformaldehyde to preserve chromatin structure .
-
Chromatin immunoprecipitation (ChIP): Use crosslinking agents like disuccinimidyl glutarate (DSG) followed by formaldehyde to stabilize histone-DNA interactions .
Key validation steps:
-
Include controls with Setd2-knockdown cells (abolishes H3K36me3) and NSD1/Ash1-depleted cells (retains H3K36me3) .
-
Verify specificity using synthetic peptides with mono-, di-, and tri-methylated H3K36 in competitive ELISA .
How does H3K36me3 localization correlate with transcriptional activity, and what experimental designs resolve conflicting data?
H3K36me3 is enriched in transcriptionally active euchromatin but paradoxically localizes to repressed heterochromatin in specific contexts (e.g., imprinted loci, pericentromeric regions) . To resolve discrepancies:
-
Stratify analysis by chromatin state: Use H3K9me3 (heterochromatin) and H3K4me3 (active promoters) as co-markers .
-
Time-course ChIP-seq: Track H3K36me3 dynamics during transcriptional bursts (e.g., EGF-induced c-Fos activation) .
-
Single-cell CUT&Tag: Resolve cell-to-cell heterogeneity in H3K36me3 distribution .
Example dataset:
Why do some studies report H3K36me3 in transcriptionally silent heterochromatin, and how can this be reconciled with its canonical role in elongation?
The presence of H3K36me3 in heterochromatin reflects context-dependent deposition mechanisms:
-
Constitutive heterochromatin: NSD methyltransferase deposits H3K36me2/3 at pericentromeric repeats independently of RNA Pol II .
-
Facultative heterochromatin: Imprinted loci retain H3K36me3 via Setd2 recruitment by non-coding RNAs .
Methodological recommendations:
-
Combine CUT&RUN (high resolution) with ATAC-seq to link H3K36me3 to chromatin accessibility .
-
Use dCas9-Setd2 fusions to ectopically deposit H3K36me3 at silent loci and monitor transcriptional leakage .
How can researchers address cross-reactivity of H3K36me3 antibodies with other methylated histone residues?
Commercial antibodies exhibit variable off-target binding:
| Antibody Clone | Cross-Reactivity | Mitigation Strategy |
|---|---|---|
| MACO0062 | H3K27me3 (15% signal in ELISA) | Pre-absorb with H3K27me3 peptide |
| MC86 | H4K20me3 (10% signal in WB) | Use Suv420h DKO cells as negative control |
Validation workflow:
-
Peptide array screening: Test antibody against 384 histone peptides .
-
Genetic knockout controls: Compare signals in Setd2−/− vs. NSD1−/− cells .
-
Dot blot quantification: Serial dilutions of recombinant methylated histones .
What explains discrepancies in reported H3K36 methyltransferase functions across studies?
NSD and Set2 methyltransferases exhibit species- and context-specific activities:
Resolution strategies:
-
Perform in vitro methyltransferase assays with purified enzymes .
-
Analyze subnuclear localization via immuno-EM (e.g., Setd2 at transcription factories) .
How to optimize ChIP protocols for H3K36me3 in low-input samples (e.g., rare cell populations)?
-
Carrier chromatin: Add 1 μg S. cerevisiae chromatin to improve precipitation efficiency .
-
Dual crosslinking: 2 mM DSG (45 min) + 1% formaldehyde (10 min) preserves fragile modifications .
-
Spike-in controls: Use Drosophila S2 chromatin with quantified H3K36me3 levels for normalization .
Quality metrics:
-
≥5% recovery in Input-adjusted calculations
-
≤2-fold variation between technical replicates
