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Histone H3. H3K27(Me3) (15-34) is a synthetic peptide derived from the C-terminal part of the histone H3 N-terminal tail (residues 15-34) of human histone H3.3, with trimethylation at lysine 27 (H3K27me3). H3K27me3 is a repressive histone modification that is associated with silenced genes and heterochromatin. It is catalyzed by the Polycomb Repressive Complex 2 (PRC2), which contains the histone methyltransferase EZH2. This mark is removed by the demethylases UTX (KDM6A) and JMJD3 (KDM6B). H3K27me3 plays a critical role in development, cell fate determination, X-chromosome inactivation, and cancer. Mutations in EZH2 and UTX are frequently found in cancers and developmental disorders. This peptide serves as a substrate for these enzymes, allowing researchers to study their activity and to screen for inhibitors. It is also used to study reader proteins that recognize H3K27me3 (e.g., Polycomb complex components, CBX proteins, and other chromodomain-containing proteins). The peptide is a research reagent for epigenetic studies.
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| ln Vitro |
In vitro, H3K27(Me3) (15-34) is used as a substrate for histone demethylases such as UTX (KDM6A) and JMJD3 (KDM6B). In a demethylase assay, the H3K27(Me3) peptide (1-10 uM) is incubated with recombinant UTX or JMJD3 in the presence of alpha-ketoglutarate, Fe(II), and ascorbate. Demethylation to H3K27me2 or H3K27me1 is monitored by mass spectrometry (MALDI-TOF or LC-MS) or by using an H3K27me3-specific antibody in an ELISA. The IC50 of inhibitors (e.g., GSK-J1, GSK-J4, GSK-J5) can be determined. Alternatively, it can serve as a substrate for the PRC2 complex (EZH2) for methylation of H3K27me0, H3K27me1, or H3K27me2 to generate H3K27me3. For methylation assays, the unmethylated or H3K27me1 peptide is incubated with PRC2 complex (purified or from cell extracts) and S-adenosylmethionine (SAM), and the incorporation of methyl groups is quantified by radiometric (3H-SAM) or by mass spectrometry. The peptide is also used as an antigen to raise H3K27me3-specific antibodies. It can be conjugated to carrier proteins (KLH, BSA) for immunization. For binding assays of "reader" proteins (e.g., CBX7, EED, or other chromodomain-containing proteins), a biotinylated version of the peptide (biotin-H3K27(Me3) (15-34)) is immobilized on streptavidin plates or magnetic beads to capture reader proteins from nuclear extracts. The TFA salt is typical and does not affect enzyme activity. The truncated peptide (15-34) includes additional residues that may contribute to proper recognition by PRC2 and other binding proteins compared to shorter peptides.
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| ln Vivo |
Not applicable (research reagent). H3K27(Me3) (15-34) is not a drug and is not administered in vivo. It is a biochemical reagent for in vitro and ex vivo studies. It can be used as a standard for mass spectrometry quantification of H3K27me3 levels in cells. It is not intended for use in animals. The product is strictly for research use in test tubes or cell-free systems.
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| Enzyme Assay |
For a non-cellular binding assay for a reader protein that recognizes H3K27me3, use a biotinylated version of the H3K27(Me3) (15-34) peptide. In a 96-well streptavidin-coated plate, add biotin-H3K27(Me3) peptide (0.1-10 uM) in binding buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20, 1 mM DTT, 0.1% BSA) and incubate for 1 hour at room temperature. Wash wells. Block with 3% BSA. Add recombinant reader protein (e.g., CBX7 chromodomain, 1-10 ug/mL) and incubate for 1-2 hours. Wash, then add primary antibody against the reader protein (or anti-His tag if His-tagged), then HRP-secondary antibody and TMB substrate. Measure absorbance at 450 nm. For competition, add increasing concentrations of unmodified H3K27(Me3) peptide or H3K27me2/me1/me0 peptides to assess specificity. For a pulldown assay, incubate biotinylated peptide with streptavidin magnetic beads, then add nuclear lysate from 293T cells. Wash, elute, and run SDS-PAGE, then Western blot for the reader protein (e.g., anti-CBX7). For a demethylase assay (cell-free): incubate H3K27(Me3) peptide (1-10 uM) with recombinant UTX or JMJD3 (0.1-1 uM) in reaction buffer (50 mM HEPES pH 7.5, 100 uM alpha-ketoglutarate, 100 uM Fe(NH4)2(SO4)2, 2 mM ascorbic acid, 1 mM DTT) at 37degC for 30-60 min. Terminate by adding 0.5% TFA. Desalt the sample and analyze by MALDI-TOF mass spectrometry. The mass shift from 2548 Da (trimethyl) to 2534 Da (dimethyl) indicates demethylation. Alternatively, use an H3K27me3-specific antibody in an ELISA: coat plate with the peptide after reaction, detect with anti-H3K27me3 antibody. For methyltransferase assays (unmethylated or H3K27me1 peptide), incubate with PRC2 complex (0.1-1 uM) and 1-10 uM 3H-SAM in reaction buffer (50 mM Tris-HCl pH 8.0, 5 mM DTT, 10 mM MgCl2) for 30-60 min. Terminate, spot on P81 filter paper, wash, and count 3H by liquid scintillation. These protocols are standard. The TFA salt is soluble in water.
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| Cell Assay |
For cell-based assays, cells (e.g., HEK293, HeLa, or cancer cell lines with high EZH2 activity) are cultured and used to isolate histones. The H3K27(Me3) (15-34) peptide is not added to cells; it is a substrate for in vitro assays. However, a cell-permeable version (with a Tat cell-penetrating peptide) could be designed to deliver the peptide into cells to compete with endogenous H3K27me3 for reader protein binding, but this is not standard. Instead, the peptide is used as a standard for mass spectrometry quantification of H3K27me3 levels in cells. For example, acid-extracted histones from cells (5-20 ug) are digested with endoproteinase Arg-C or trypsin. A known amount of stable isotope-labeled H3K27(Me3) peptide (e.g., 13C/15N-labeled) is added to the digest as an internal standard. The mixture is analyzed by LC-MS/MS in selected reaction monitoring (SRM) mode. The ratio of the endogenous (unlabeled) peptide to the internal standard allows absolute quantification of H3K27me3 levels. This method is used to measure changes in H3K27me3 occupancy after drug treatment (e.g., EZH2 inhibitors) or in disease models. For a ChIP control, the unlabeled peptide is not used. For a competition assay in vitro, as described above. Cell-based assays are not performed with this peptide because it is not cell-permeable. All experiments using isolated histones or nuclear extracts can be performed. The TFA salt is acceptable; dissolve in water or 0.1% TFA to 1-10 mM stock. Use in buffer compatible with enzymatic assays.
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| Animal Protocol |
Not applicable. H3K27(Me3) (15-34) is not administered to animals. There are no in vivo animal experiment protocols for this peptide because it is an in vitro research reagent. It can be used ex vivo: e.g., brain tissue from a mouse is homogenized, histones are extracted, and the peptide is used as a mass spectrometry standard for quantification of H3K27me3. However, that is an analytical procedure, not an animal procedure. The product is for research use only, not for animal dosing.
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| ADME/Pharmacokinetics |
No pharmacokinetic (PK) data are available for H3K27(Me3) (15-34). This peptide is not administered in vivo; therefore, PK parameters (absorption, distribution, metabolism, excretion, half-life) are not relevant. The peptide is for in vitro use only.
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| Toxicity/Toxicokinetics |
No toxicity data are available for H3K27(Me3) (15-34). As a synthetic peptide composed of naturally occurring L-amino acids, it is generally considered to have low toxicity. In vitro, the peptide at concentrations up to 100 uM does not interfere with enzymatic assays and is not cytotoxic to cells when added to culture media (though it is not cell-permeable). However, because it is not intended for in vivo use, toxicology studies are not conducted. The TFA salt is present in low, stoichiometric amounts and is not toxic. Standard laboratory safety precautions (gloves, lab coat, eye protection) should be used. The product is for research use only and is not intended for human or animal use.
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| References |
[1]. Elizabeth T Wiles, et al. H3K27 Methylation: A Promiscuous Repressive Chromatin Mark. Curr Opin Genet Dev. 2017 Apr;43:31-37.
[2]. Brian P Chadwick, et al. Multiple Spatially Distinct Types of Facultative Heterochromatin on the Human Inactive X Chromosome. Proc Natl Acad Sci U S A. 2004 Dec 14;101(50):17450-5. |
| Additional Infomation |
H3K27 trimethylation (H3K27me3) is a repressive histone modification that is essential for gene silencing, Polycomb-mediated repression, development, and maintenance of cell identity. It is catalyzed by the histone methyltransferase EZH2 (Enhancer of Zeste Homolog 2), a component of the Polycomb Repressive Complex 2 (PRC2). H3K27me3 is removed by the demethylases UTX (KDM6A) and JMJD3 (KDM6B). Dysregulation of H3K27me3 is implicated in numerous cancers (e.g., lymphoma, breast cancer, prostate cancer) and developmental disorders (e.g., Kabuki syndrome). The H3K27(Me3) (15-34) peptide is a synthetic peptide that includes residues 15-34 of human histone H3 with trimethylated lysine 27. It is longer than the commonly used 1-20 peptide, which may be important for proper recognition by PRC2 and certain reader proteins. This peptide is used as an in vitro substrate for methyltransferases and demethylases, as a standard for mass spectrometry, and as a tool for studying reader proteins. The TFA salt is used for solubility and storage. This product is not a drug and has no therapeutic use; it is a research-grade chemical for epigenetic studies.
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| Molecular Formula |
C87H157N30O25
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| Molecular Weight |
2023.40
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| Appearance |
White to off-white solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
H2O :≥ 50 mg/mL (~24.71 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.4942 mL | 2.4711 mL | 4.9422 mL | |
| 5 mM | 0.0988 mL | 0.4942 mL | 0.9884 mL | |
| 10 mM | 0.0494 mL | 0.2471 mL | 0.4942 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Products are for research use only; We do not sell to patients
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