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Histone H3. H3K4(Me2) (1-20) is a synthetic peptide corresponding to the N-terminal tail (amino acids 1-20) of human histone H3.1, with dimethylation at lysine 4 (H3K4me2). H3K4me2 is an epigenetic mark associated with active gene transcription. It is found in euchromatin (transcriptionally active regions) and is enriched at enhancer regions and at the promoter-proximal regions of active genes. H3K4me2 is recognized by reader proteins containing PHD fingers or Tudor domains (e.g., CHD1, BPTF, ING2). The methylation is catalyzed by histone methyltransferases (e.g., SET1/MLL complexes, SETD1A/B, MLL1-4) and removed by demethylases (e.g., KDM5 family, LSD1). H3K4me2 is an intermediate in the dynamic methylation cycle, and its levels are regulated in response to cellular stress, including DNA damage. Specifically, H3K4me2 regulates the recovery of protein biosynthesis and homeostasis following DNA damage. This peptide is used as an in vitro substrate for these enzymes. It is not a drug; it is a research reagent for studying chromatin biology and gene regulation.
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| ln Vitro |
In vitro, H3K4(Me2) (1-20) is used as a substrate for histone demethylases such as KDM5A (JARID1A) and KDM5B (JARID1B), as well as for methyltransferases. In a demethylase assay, the H3K4(Me2) peptide (1-10 uM) is incubated with recombinant KDM5A in the presence of alpha-ketoglutarate, Fe(II), and ascorbate. Demethylation to monomethylated (H3K4me1) or unmethylated (H3K4me0) forms is monitored by mass spectrometry (MALDI-TOF or LC-MS) or by using an H3K4me2-specific antibody in an ELISA. The IC50 of inhibitors can be determined. Alternatively, it can serve as a substrate for lysine methyltransferases (e.g., MLL1 complex, SETD1A) to generate H3K4me2 or H3K4me3. For methylation assays, the monomethylated H3K4 peptide or unmethylated peptide is incubated with a methyltransferase 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 H3K4me2-specific antibodies. It can be conjugated to carrier proteins for immunization. It is also used in competition ELISAs to confirm antibody specificity. For binding assays of "reader" proteins (e.g., CHD1), a biotinylated version of the peptide 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.
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| ln Vivo |
Not applicable (research reagent). H3K4(Me2) (1-20) 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 H3K4me2 levels in cell extracts. 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 H3K4me2, use a biotinylated version of the H3K4(Me2) (1-20) peptide. In a 96-well streptavidin-coated plate, add biotin-H3K4(Me2) 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., CHD1, 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 H3K4(Me2) peptide or H3K4me3/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-CHD1). For a demethylase assay (cell-free): incubate H3K4(Me2) peptide (1-10 uM) with recombinant KDM5A (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 1142 Da (dimethyl) to 1128 Da (monomethyl) indicates demethylation. Alternatively, use an H3K4me2-specific antibody in an ELISA: coat plate with the peptide after reaction, detect with anti-H3K4me2 antibody. For methyltransferase assays (unmethylated or H3K4me1 peptide), incubate with methyltransferase (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) are cultured and used to isolate histones. The H3K4(Me2) (1-20) 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 H3K4me2 for reader protein binding, but this is not standard. Instead, the peptide is used as a standard for mass spectrometry quantification of H3K4me2 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 H3K4(Me2) 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 H3K4me2 levels. This method is used to measure changes in H3K4me2 occupancy after drug treatment 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. H3K4(Me2) (1-20) 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 H3K4me2. 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 H3K4(Me2) (1-20). 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 H3K4(Me2) (1-20). 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]. Mulder KW, et al. Regulation of histone H3K4 tri-methylation and PAF complex recruitment by the Ccr4-Not complex. Nucleic Acids Res. 2007;35(7):2428-2439.
[2]. Wang S, et al. H3K4me2 regulates the recovery of protein biosynthesis and homeostasis following DNA damage. Nat Struct Mol Biol. 2020;27(12):1165-1177. |
| Additional Infomation |
H3K4 dimethylation (H3K4me2) is a histone post-translational modification (PTM) that is associated with active gene transcription. It is highly enriched at enhancer regions and promoter-proximal regions, and its levels are often co-regulated with H3K4me3 and H3K4me1. H3K4me2 plays a role in the recovery of protein biosynthesis and homeostasis following DNA damage. The H3K4(Me2) (1-20) peptide is a synthetic version of the N-terminal tail of histone H3 (residues 1-20) with dimethylated lysine 4. It 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 |
C93H171N35O27
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| Molecular Weight |
2211.61
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| Appearance |
Typically exists as solid at room temperature
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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 :~25 mg/mL (~11.30 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 | 0.4522 mL | 2.2608 mL | 4.5216 mL | |
| 5 mM | 0.0904 mL | 0.4522 mL | 0.9043 mL | |
| 10 mM | 0.0452 mL | 0.2261 mL | 0.4522 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.
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