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| Targets |
MHC class I H-2Db. Gp100 (25-33), mouse is an MHC class I-restricted peptide epitope derived from the melanocyte-specific protein gp100 (Pmel17). In mice, this 9-mer peptide (EGSRNQDWL) binds to the MHC class I molecule H-2Db (mouse MHC class I allele). The peptide-MHC complex is presented on the surface of antigen-presenting cells (APCs) and recognized by CD8+ cytotoxic T lymphocytes (CTLs). Gp100 (25-33) is a "self/tumor antigen" because it is expressed in normal melanocytes (pigment-producing cells) but is also highly expressed in melanoma cells. This peptide does not target a receptor or enzyme; rather, it is an antigen that, when presented on MHC, can activate specific T cells. It is used to study T cell recognition, activation, and tolerance in melanoma models.
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| ln Vitro |
In vitro, Gp100 (25-33), mouse TFA is used to stimulate specific CD8+ T cell clones or T cell hybridomas in culture. The peptide (at concentrations of 0.1-10 uM) is presented by H-2Db-expressing antigen-presenting cells (such as dendritic cells or the T2-Db cell line) to gp100-specific T cells. T cell activation is measured by production of cytokines (IFN-gamma, IL-2, TNF-alpha) by ELISA or ELISpot, upregulation of activation markers (CD69, CD25) by flow cytometry, or proliferation (3H-thymidine incorporation). The peptide is also used in tetramer staining: MHC class I H-2Db molecules are tetramerized with the Gp100 (25-33) peptide and fluorescently labeled; these tetramers bind specifically to T cell receptors (TCR) on Gp100-specific CD8+ T cells, allowing quantification of antigen-specific T cells by flow cytometry. The peptide does not have direct enzymatic or receptor-modulating activity; its activity is strictly immune-mediated. The TFA salt does not affect peptide presentation. The peptide's EC50 for T cell activation typically ranges from 0.1-10 nM to 1 uM, depending on the T cell clone and assay.
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| ln Vivo |
In vivo, Gp100 (25-33), mouse TFA is used to study anti-tumor immune responses in mouse models of melanoma. In B16-F10 mouse melanoma models (which express the endogenous gp100 antigen), immunization with Gp100 (25-33) peptide (administered with adjuvant such as complete Freund's adjuvant (CFA), CpG, or in dendritic cell vaccines) induces a specific CD8+ T cell response against gp100-expressing tumor cells. For example, C57BL/6 mice are immunized subcutaneously with 50-100 ug of Gp100 peptide emulsified in CFA or mixed with CpG (50 ug) and IFA. After 7-14 days, splenocytes are harvested, and T cell responses are analyzed by IFN-gamma ELISpot or intracellular cytokine staining (ICS). In tumor challenge experiments, mice are immunized with the peptide (with adjuvant) on day -7 and day -3, then challenged with B16-F10 melanoma cells (5 × 10^5 cells subcutaneously) on day 0. Peptide immunization reduces tumor growth and can prolong survival compared to unimmunized mice. However, because gp100 is a self-antigen, the T cell response may be tolerized, and the efficacy of gp100 peptide vaccines is limited without checkpoint inhibitors. Combination with anti-CTLA-4 or anti-PD-1 antibodies enhances anti-tumor immunity. Gp100 (25-33) peptide is also used in adoptive T cell transfer (ACT) models: gp100-specific T cells are expanded in vitro using the peptide and transferred into tumor-bearing mice. The peptide is also used to generate T cell hybridomas. The mouse sequence (EGSRNQDWL) is distinct from the human gp100 25-33 epitope (KVPRNQDWL). Therefore, the mouse peptide is specific for mouse MHC H-2Db and is used only in mouse studies. The TFA salt is suitable for in vivo injection; the peptide is dissolved in PBS or DMSO and diluted in PBS before injection. The peptide is well-tolerated at doses up to 200 ug per mouse.
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| Enzyme Assay |
For direct binding assays (non-cellular), the binding affinity of the Gp100 (25-33) mouse peptide to the H-2Db MHC class I molecule can be measured using an MHC-peptide binding assay. Recombinant H-2Db protein (with beta2-microglobulin) is used. A fluorescence polarization (FP) or ELISA-based competition assay is standard. For FP: Purified H-2Db (0.1-1 uM) is incubated with a fluorescently labeled reference peptide (e.g., FITC-labeled H-2Db-binding peptide) in binding buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% NP-40, 1 mM DTT). Varying concentrations of unlabeled Gp100 (25-33) mouse peptide (0.001-1000 uM) are added, and the mixture is incubated for 24-48 hours at room temperature to allow peptide binding and exchange. Fluorescence polarization is measured (excitation 485 nm, emission 520 nm). The IC50 (concentration of unlabeled peptide that displaces 50% of the reference peptide) is determined. The IC50 or Kd can be reported. For an ELISA-based assay: 96-well plates are coated with H-2Db/beta2m complex. A known biotinylated H-2Db-binding peptide is added with varying concentrations of unlabeled Gp100 (25-33) mouse peptide; binding is detected with streptavidin-HRP and TMB. The IC50 is calculated. Alternatively, a surface plasmon resonance (SPR) assay can be performed: Immobilized H-2Db/beta2m protein on a sensor chip; flow the peptide at 0.1-1000 uM; determine KD. The affinity of this peptide for H-2Db is in the low micromolar to high nanomolar range. For peptide stability or degradation assays: Incubate the peptide in mouse serum (37degC for 0-24 h), then analyze by HPLC or LC-MS to measure half-life.
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| Cell Assay |
For cell-based T cell activation assays, splenocytes or purified CD8+ T cells from gp100-specific T cell receptor (TCR) transgenic mice (e.g., pmel-1 mice) are used. Alternatively, a T cell hybridoma expressing a gp100-specific TCR is used. For the ELISpot assay: 96-well ELISpot plates are coated with anti-IFN-gamma antibody overnight. Splenocytes from immunized mice (or from pmel-1 mice) are plated at 1-2 × 10^5 cells/well in RPMI-1640 with 10% FBS. Gp100 (25-33) mouse peptide is added at serial dilutions (0.001-1000 nM). H-2Db-expressing antigen-presenting cells (e.g., T2-Db cells, 1 × 10^4 cells/well) may be added as feeders if splenocytes are not used. After 20-24 hours at 37degC, cells are washed, and biotinylated anti-IFN-gamma detection antibody is added, followed by streptavidin-AP, and spots are developed with BCIP/NBT. Spots are counted using an ELISpot reader. For cytokine ELISA: Splenocytes (2 × 10^5 cells/well) are stimulated with peptide (0.01-1000 nM) in 96-well plates for 48-72 hours. Supernatants are collected, and IFN-gamma, IL-2, or TNF-alpha are measured by ELISA. For flow cytometry-based T cell activation: Splenocytes are stimulated with peptide (0.1-10 uM) for 6-18 hours in the presence of brefeldin A (last 4-6 hours). Cells are surface-stained for CD8, then fixed, permeabilized, and stained for intracellular IFN-gamma or IL-2. The percentage of IFN-gamma+ CD8+ T cells is measured. For proliferation assays: CFSE-labeled splenocytes are stimulated with peptide (0.01-10 uM) for 72-96 hours; dilution of CFSE in CD8+ T cells is analyzed by flow cytometry. For tetramer staining: PE-labeled H-2Db/Gp100 (25-33) tetramers are used to stain splenocytes from immunized mice. Cells are stained with tetramer (1-2 ug/mL) for 30 min at room temperature, then with anti-CD8 antibody, and analyzed by flow cytometry. All experiments should include an irrelevant control peptide (e.g., OVA 257-264) as a negative control. The TFA salt is dissolved in DMSO (10 mM stock) or water; avoid repeated freeze-thaw cycles. Peptide purity ≥95% is critical for accurate results.
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| Animal Protocol |
For in vivo immunization studies, female C57BL/6 mice (6-8 weeks old) are used. Gp100 (25-33), mouse TFA is dissolved in sterile PBS (or DMSO then diluted in PBS) at a concentration of 1 mg/mL. For active immunization, the peptide is emulsified with an adjuvant. For CFA/IFA: Mix an equal volume of peptide solution (50-100 ug peptide per mouse) with complete Freund's adjuvant (CFA) for primary immunization, or with incomplete Freund's adjuvant (IFA) for booster immunizations. Emulsify by mixing with a syringe until a stable water-in-oil emulsion forms (a drop of emulsion does not disperse in water). Inject 100 uL emulsion subcutaneously (s.c.) at the base of the tail or in the flank. For CpG-based immunization: Mix peptide (50-100 ug) with CpG ODN (50 ug) in PBS (total volume 100 uL) and inject s.c. or i.p. A typical schedule: Day 0: primary immunization (peptide + CFA). Day 7 or 14: booster immunization (peptide + IFA). On day 14-21, mice are euthanized, and spleens are harvested for T cell assays (ELISpot, ICS, tetramer). For tumor challenge experiments: Mice are immunized on day -14 and day -7 (with CFA/IFA or CpG). On day 0, B16-F10 melanoma cells (5 × 10^5 in 100 uL PBS) are injected subcutaneously into the right flank. Tumor volume is measured with calipers every 2-3 days. Mice are monitored for survival. For combination therapy: Anti-PD-1 antibody (200 ug, i.p.) or anti-CTLA-4 antibody (100 ug, i.p.) is administered on day 0, 3, 6, 9 after tumor challenge. For adoptive T cell transfer (ACT): CD8+ T cells are isolated from pmel-1 mice, activated in vitro with peptide (1 uM) and IL-2 (30 IU/mL) for 5 days, then expanded. These cells (5 × 10^6 in 200 uL PBS) are adoptively transferred intravenously into tumor-bearing mice (day 7 after B16-F10 challenge). The peptide is also used for expanding T cells in vitro. The TFA salt is acceptable for in vivo use; however, ensure that the final solution pH is between 6-8. All animal procedures must be approved by IACUC.
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| ADME/Pharmacokinetics |
No detailed pharmacokinetic data are available for Gp100 (25-33), mouse TFA. As a 9-amino acid peptide (MW ~1.2 kDa), it is highly water-soluble and will be rapidly cleared from circulation when administered intravenously or subcutaneously. In vivo, after subcutaneous injection, the peptide is likely absorbed slowly from the injection site (especially when emulsified in CFA/IFA). However, the peptide is intended to act as an antigen to be taken up by antigen-presenting cells (APCs) at the injection site, not to have systemic PK. Free peptide in the circulation is rapidly degraded by proteases and cleared by the kidneys (t1/2 < 10 min). The TFA salt does not alter PK properties. For immunization studies, the peptide is administered in adjuvant, which forms a depot at the injection site, releasing the peptide slowly over days to weeks. Therefore, the PK of the free peptide is not relevant to its immunogenicity. The peptide is not a drug; detailed PK parameters are not published.
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| Toxicity/Toxicokinetics |
No specific toxicity data are available for Gp100 (25-33), mouse TFA. As a 9-amino acid peptide derived from a self/tumor antigen, it is generally well-tolerated. In vivo, C57BL/6 mice immunized with the peptide (50-100 ug per mouse in CFA/IFA) show no overt signs of toxicity (no weight loss, no behavioral changes, no mortality). Injection site reactions (swelling, erythema) can occur due to the adjuvant (CFA), not the peptide. Systemic cytokine release (IFN-gamma, IL-2) is part of the intended immune response, not toxicity. Some mice may develop autoimmune vitiligo (depigmentation of hair/fur) due to immune attack on normal melanocytes, which is an on-target autoimmune effect, not a toxicological hazard. This model is used to study autoimmune side effects of cancer immunotherapies. No genotoxicity, organ toxicity, or carcinogenicity has been reported. The TFA salt is present in low amounts and is considered non-toxic. The peptide is for research use only; it is not approved for human or veterinary use. Standard laboratory safety precautions (gloves, lab coat) should be used. Unlike the human peptide, the mouse peptide is used only in mouse models and is not intended for human therapy.
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| References |
[1]. Gal Cafri, et al. Production of LacZ inducible T cell hybridoma specific for human and mouse gp100 25-33 peptides. PLoS One. 2013;8(2):e55583.
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| Additional Infomation |
The gp100 (Pmel17) protein is a melanocyte-specific glycoprotein involved in melanosome biogenesis and pigmentation. It is highly expressed in melanoma cells and has been extensively studied as a target for cancer immunotherapy, including vaccines, adoptive T cell transfer (ACT), and checkpoint inhibitors. The gp100 25-33 epitope in mice (EGSRNQDWL) is restricted by H-2Db and was identified as a dominant CD8+ T cell epitope in B16-F10 melanoma-bearing mice. The corresponding human epitope is gp100 209-217 (ITDQVPFSV) or 280-288 (YLEPGPVTA), which are HLA-A2-restricted. The mouse peptide is used as a tool to study melanoma immunology in the well-established B16-F10 model. The TFA salt is used to improve solubility. The peptide is for research use only; it is not an approved drug and has not been tested in human subjects. This product is not intended for therapeutic use.
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| Molecular Formula |
C48H70F3N15O19
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| Molecular Weight |
1218.15
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| Related CAS # |
Gp100 (25-33), mouse;212370-36-0
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 0.8209 mL | 4.1046 mL | 8.2092 mL | |
| 5 mM | 0.1642 mL | 0.8209 mL | 1.6418 mL | |
| 10 mM | 0.0821 mL | 0.4105 mL | 0.8209 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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