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| 1mg |
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| Targets |
(Arg)9, FAM-labeled is used as a tool to study cellular uptake mechanisms, intracellular trafficking, and subcellular localization of cell-penetrating peptides and their cargoes. The FAM fluorophore allows tracking of the peptide's binding to the cell surface, endocytosis, and intracellular distribution. (Arg)9 is known to bind to heparan sulfate proteoglycans (HSPGs) on the cell surface, which facilitates its internalization primarily through macropinocytosis and other endocytic pathways.
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| ln Vitro |
In vitro, (Arg)9 peptides are internalized by a wide variety of cell types, including primary cells and cell lines. The FAM label allows quantitative measurement of cellular uptake by flow cytometry (median fluorescence intensity) or by fluorescence microscopy. The peptide typically shows rapid uptake (within 15-60 minutes) in a concentration-dependent manner (0.1-10 uM). The uptake of (Arg)9 is inhibited by low temperature, depletion of cellular ATP, and treatment with macropinocytosis inhibitors such as EIPA and rottlerin, confirming energy-dependent, macropinocytosis-mediated entry.
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| ln Vivo |
In vivo, (Arg)9 peptides accumulate in various tissues following systemic administration. For imaging studies, mice are injected intravenously (IV) or intraperitoneally (IP) with (Arg)9, FAM-labeled at doses of 0.5-5 mg/kg. The FAM fluorescence (λ_ex 494 nm, λ_em 518 nm) can be detected in tissue sections by fluorescence microscopy or whole-body imaging. The peptide distributes primarily to the kidneys (due to renal clearance), liver, and lungs, and also shows some brain uptake. (Arg)9 is often conjugated to therapeutic cargoes to enhance their cellular delivery and tissue penetration in vivo.
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| Enzyme Assay |
A FAM fluorescence-based flow cytometry uptake assay is performed. Adherent cells (e.g., HeLa, CHO, HEK293) are seeded in 12- or 24-well plates. When cells reach 70-80% confluence, they are treated with (Arg)9, FAM-labeled at concentrations of 0.1-10 uM for 15-120 minutes at 37degC. Following treatment, cells are washed 3× with PBS to remove unbound peptide, trypsinized, and resuspended in FACS buffer. Fluorescence is measured using a flow cytometer (488 nm excitation, 525/530 nm emission). Median fluorescence intensity is recorded and normalized to cell count. For inhibition studies, cells are pre-incubated with uptake inhibitors (EIPA, cytochalasin D, rottlerin, nystatin) for 30 minutes.
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| Cell Assay |
For confocal microscopy, cells are seeded on coverslips or in glass-bottom dishes. Cells are treated with (Arg)9, FAM-labeled (1-5 uM) for 30-60 minutes, washed, and fixed with 4% paraformaldehyde (PFA). Nuclei are counterstained with DAPI or Hoechst 33342. For endosomal/lysosomal localization, cells are co-stained with LysoTracker Red DND-99 (50 nM) or CellLight Early Endosomes-GFP (BacMam 2.0). For plasma membrane staining, cells are stained with wheat germ agglutinin (WGA) Alexa Fluor 647. Images are acquired using a confocal laser scanning microscope and analyzed using ImageJ/Fiji software.
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| Animal Protocol |
For in vivo biodistribution studies, female BALB/c mice (6-8 weeks) are injected intravenously (IV) via the tail vein or intraperitoneally (IP) with (Arg)9, FAM-labeled (1-10 mg/kg) in sterile saline. At various time points post-injection (0.5, 1, 2, 4, 8, 12, 24 hours), mice are euthanized, and organs (liver, kidney, spleen, lung, heart, brain, intestine) are collected. For fluorescence imaging, tissues are imaged ex vivo using an IVIS Spectrum imaging system (λ_ex 465 nm, λ_em 520 nm). Alternatively, tissues are frozen, sectioned (10-20 um), and imaged by fluorescence microscopy. For quantitative biodistribution, tissues are homogenized and fluorescence intensity is measured by plate reader.
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| ADME/Pharmacokinetics |
(Arg)9, FAM-labeled is a cell-penetrating peptide (CPP) tool. In vivo, the peptide distributes primarily to the kidneys due to its small size (MW ~1500 g/mol for (Arg)9 plus FAM) and cationic nature, leading to glomerular filtration and renal clearance. The plasma half-life is short (typically 5-20 minutes in mice) due to rapid renal clearance. For PK analysis, blood samples are collected at various time points, plasma is separated, and FAM fluorescence is measured. For extended circulation, (Arg)9 is often conjugated to larger carriers (e.g., PEG, proteins, or nanoparticles).
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| Toxicity/Toxicokinetics |
As a peptide composed of natural amino acids (L-arginine) with a FAM dye, (Arg)9, FAM-labeled is generally non-toxic at the concentrations used for cell uptake studies (1-20 uM). In cell viability assays (MTT, CellTiter-Glo), no significant cytotoxicity is observed up to 20 uM. At very high concentrations (>50 uM), some membrane disruption and cytotoxicity may occur due to the cationic nature of the peptide. In vivo, doses up to 10-20 mg/kg are well-tolerated in mice with no significant weight loss, organ toxicity, or behavioral changes. No genotoxicity or immunogenicity is expected.
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| References |
[1]. CPProtectides: Rapid uptake of well-folded β-hairpin peptides with enhanced resistance to intracellular degradation. Pept Sci (Hoboken). 2019 Mar; 111(2): e24092.
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| Additional Infomation |
Cell-penetrating peptides (CPPs) including (Arg)9 are widely used as delivery vehicles for a variety of therapeutic and imaging cargoes, including oligonucleotides (siRNA, antisense, miRNA), proteins, peptides, and nanoparticles. The nona-arginine sequence (Arg9) is one of the most efficient CPPs and is often used as a positive control in CPP uptake studies. The FAM label (5-carboxyfluorescein) is a bright, photostable green fluorescent dye with λ_ex 494 nm, λ_em 518 nm, and is commonly used for flow cytometry and fluorescence microscopy. This product is for research use only and is not FDA-approved for human therapy.
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| Molecular Formula |
C75H120N36O16
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| Molecular Weight |
1782.00
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| Appearance |
Brown to reddish brown 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) |
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 | 0.5612 mL | 2.8058 mL | 5.6117 mL | |
| 5 mM | 0.1122 mL | 0.5612 mL | 1.1223 mL | |
| 10 mM | 0.0561 mL | 0.2806 mL | 0.5612 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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