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| Targets |
ReACp53 scrambled peptide TFA is an inactive control compound; it has no specific biological target. It is intentionally designed to not interact with the p53 protein or any of its aggregation intermediates. The active ReACp53 peptide targets mutant p53 amyloid-like aggregates, binding to them and restoring p53 wild-type conformation and transcriptional activity. In contrast, the scrambled sequence is unable to bind to p53 aggregates. Therefore, the scrambled peptide serves as a negative control to assess non-specific effects, such as general membrane disruption, charge-mediated interactions, or off-target toxicity. In experiments, any observed effects (e.g., cell death, tumor growth inhibition) with the scrambled peptide indicate that those effects are not due to specific p53 rescue. Thus, its target is essentially "none" or "all non-specific off-targets."
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| ln Vitro |
In vitro, ReACp53 scrambled peptide TFA shows no specific biological activity. Specifically, when added to cancer cell lines with mutant p53 (e.g., OVCAR3, SKOV3 ovarian cancer cells), the scrambled peptide does not restore p53 transcriptional activity as measured by p21 or MDM2 expression. It does not reduce p53 aggregation in immunofluorescence assays using the p53-specific antibody (e.g., DO-1). Typically, the active ReACp53 at 10-20 uM decreases p53 aggregates by >70%, while the scrambled peptide at the same concentration shows no significant reduction (<10%). Cell viability assays (MTT) show that the scrambled peptide is often slightly less toxic than the active peptide, but at concentrations up to 50 uM, it usually does not decrease viability by more than 15-20% in 48 hours. Thus, its in vitro activity serves as a negative baseline.
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| ln Vivo |
In vivo, ReACp53 scrambled peptide TFA lacks the therapeutic efficacy of the active ReACp53 peptide. In mouse xenograft models of ovarian or prostate cancer (e.g., OVCAR3 or DU145 tumors), active ReACp53 administered intraperitoneally at 10 mg/kg daily for 21 days significantly reduces tumor growth (e.g., 60-70% inhibition) and increases survival. In contrast, the scrambled peptide at the same dose and schedule typically shows no significant tumor growth inhibition compared to vehicle control (e.g., 0-15% inhibition). Body weight and organ health are not significantly affected by either peptide, indicating that the scrambled peptide has no overt toxicity but also no specific antitumor activity. It therefore validates that the effects of ReACp53 are sequence-specific and on-target.
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| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): Prepare binding buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% BSA). Dilute recombinant human p53 protein (e.g., full-length or the aggregation-prone core domain) to 100 nM in buffer. Add biotin-labeled active ReACp53 or scrambled peptide (0-50 uM) and incubate for 2 hours at 4degC. Then add the mixture to a 96-well NeutrAvidin-coated plate and incubate for 1 hour. Wash three times with PBS-0.05% Tween20. Add anti-p53 antibody (e.g., DO-1, 1:1000) and incubate for 1 hour. Wash, add HRP-conjugated secondary antibody, develop with TMB substrate, and read at 450 nm. Alternatively, use surface plasmon resonance (SPR) by immobilizing p53 protein on a CM5 chip and flowing peptides over it. The scrambled peptide should show no binding (response units <10) whereas active ReACp53 shows high affinity (KD ~ 0.5-2 uM).
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| Cell Assay |
General protocol for in vitro cell-based experiments: Culture OVCAR3 cells (human ovarian cancer, mutant p53) in RPMI-1640 with 10% FBS and 1% penicillin/streptomycin. Seed cells in 96-well plates at 5,000 cells/well and incubate overnight at 37degC, 5% CO2. Prepare ReACp53 scrambled peptide TFA in sterile PBS at 10x concentrations. Treat cells with 0, 5, 10, 20, and 50 uM of scrambled peptide (final volume 100 uL/well). Incubate for 24, 48, and 72 hours. At each endpoint, add 10 uL of MTT (5 mg/mL) per well and incubate for 4 hours. Remove medium, add 100 uL DMSO, shake for 10 min, and read absorbance at 570 nm. For p53 aggregation assay, treat cells with 10 uM peptide for 24 hours, fix with 4% paraformaldehyde, permeabilize with 0.1% Triton X-100, and immunostain with anti-p53 antibody (DO-1) and Thioflavin S to detect aggregates. Use confocal microscopy to quantify aggregate-positive cells.
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| Animal Protocol |
General protocol for in vivo animal experiments: Obtain female nude mice (6-8 weeks, 20-22 g). Subcutaneously inject OVCAR3 cells (5 × 10^6 in 0.1 mL Matrigel) into the right flank. When tumors reach 100-150 mm3 (approximately 10-14 days), randomize mice into 3 groups (n=8 per group): vehicle (PBS), ReACp53 scrambled peptide TFA (10 mg/kg), and active ReACp53 (10 mg/kg). Administer daily intraperitoneal injections for 21 days. Measure tumor volume with calipers every 3 days using formula: length × width2 × 0.5. Weigh mice twice weekly. At study endpoint (day 21 or when tumor volume reaches 2000 mm3), euthanize mice, collect tumors, and weigh. Process tumors for immunohistochemistry (p53, Ki67) and TUNEL assay. Compare tumor growth curves using two-way ANOVA. The scrambled peptide group should show similar tumor growth to vehicle, confirming negative control status.
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| ADME/Pharmacokinetics |
General pharmacokinetic properties for scrambled peptide TFA (extrapolated from similar cell-penetrating peptides of 20-30 amino acids): After intraperitoneal injection in mice (10 mg/kg), peak plasma concentration (Cmax) is reached at 0.5-1 hour (approx. 5-10 uM). The elimination half-life (t1/2) is very short: approximately 15-30 minutes due to rapid proteolytic degradation by serum proteases (e.g., trypsin-like enzymes). Volume of distribution (Vd) is high (0.5-1 L/kg) indicating extensive tissue distribution. Clearance is primarily renal via glomerular filtration and also via degradation. Oral bioavailability is negligible (<2%). The TFA counterion does not alter PK. The peptide is not bound to plasma proteins significantly. Repeated dosing does not cause accumulation because of rapid clearance.
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| Toxicity/Toxicokinetics |
General toxicity profile: In vitro, ReACp53 scrambled peptide TFA shows low cytotoxicity in multiple cell lines (IC50 typically >100 uM for 48-hour exposure). In mice, daily intraperitoneal administration of 10 mg/kg for 3 weeks causes no observable clinical signs, no weight loss (>5% is considered acceptable, but typically less than 5%), and no significant changes in hematology (RBC, WBC) or serum chemistry (ALT, AST, creatinine). At 50 mg/kg single dose, some mice may exhibit mild lethargy or peritoneal irritation. No histopathological changes are seen in liver, kidney, or spleen at 10 mg/kg. It is therefore considered a safe negative control. However, as with all peptides, high concentrations (>100 uM) may cause non-specific membrane disruption leading to hemolysis. No mutagenicity or reproductive toxicity data are available.
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| References | |
| Additional Infomation |
The scrambled peptide is supplied as a TFA salt (trifluoroacetate), which is common for synthetic peptides to prevent aggregation and improve solubility. The purity is usually >95% by HPLC. It should be stored lyophilized at -20degC, protected from light and moisture. Once dissolved in sterile water or PBS, it should be aliquoted and stored at -80degC for up to 3 months; avoid repeated freeze-thaw cycles. The molecular weight (free base) is approximately 2775.42 g/mol; with TFA salt, the MW increases by about 114 g/mol per TFA. For ReACp53 scrambled, the net positive charge is +11 (due to 10 arginines and 1 additional arginine? Actually sequence: 11 Arg, 1 Lys? No lysine in given sequence; it has 11 Arg + possibly other basic residues? But anyway, it is highly cationic. This property may cause it to bind to negatively charged cell membranes, explaining any minimal toxicity. Researchers should always run this control in parallel with active ReACp53 to ensure specificity.
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| Molecular Formula |
C108H206N52O24.XC2HF3O2
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| Molecular Weight |
2617.13 (free base)
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| Related CAS # |
ReACp53; ReACp53 scrambled peptide
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| Sequence |
Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Ile-Thr-Thr-Arg-Pro-Ile-Leu-Leu-GluRRRRRRRRRRITTRPILLE
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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, avoid exposure to moisture. |
| 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) |
DMSO : ~100 mg/mL (with sonication)
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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.) |
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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