| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Targets |
The NLS peptide acts as a ligand for the nuclear import receptor Importin alpha. In the cytoplasm, the NLS sequence binds directly to the importin alpha subunit of the importin alpha/beta heterodimer with high affinity. This binding triggers the transport of the cargo-NLS complex through the nuclear pore complex (NPC) via a RanGTP-dependent mechanism. Once inside the nucleus, the binding of RanGTP to importin beta causes the disassembly of the import complex, releasing the NLS-tagged cargo into the nucleoplasm. The importins are then recycled back to the cytoplasm for another round of transport. This specific interaction and the subsequent nuclear translocation cascade are essential for the function of many nuclear proteins. The NLS peptide can be chemically conjugated to non-native cargos to artificially drive their nuclear localization.
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| ln Vitro |
In vitro, the NLS peptide has been shown to dramatically enhance the nuclear accumulation of conjugated cargos in cell-based assays. In studies using permeabilized cells or live-cell imaging, the addition of an NLS peptide (typically at concentrations of 10-50 uM) can increase the nuclear import rate of a fluorescently labeled protein cargo by 5- to 10-fold. It has been extensively utilized to enhance the efficiency of genome editing tools, such as CRISPR-Cas9. For instance, fusing multiple copies of the SV40 NLS to the Cas9 protein significantly improves its nuclear localization and, consequently, its gene-editing activity. This approach is now a standard feature in the design of Cas9 vectors for genome engineering.
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| ln Vivo |
While the NLS peptide itself is not a therapeutic drug, its in vivo applications are numerous in the context of research. When conjugated to plasmid DNA, proteins, or nanoparticles, the NLS has been shown to improve the nuclear delivery of these cargos in living animals. In xenograft tumor models, systemic administration of NLS-conjugated nanoparticles has resulted in enhanced accumulation of the payload within the nucleus of tumor cells, which is often a critical barrier for the efficacy of gene-based therapies. The NLS peptide, however, is rapidly cleared from circulation when administered alone. Its primary in vivo utility is as a functional domain on larger, more stable constructs.
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| Enzyme Assay |
The interaction between the NLS peptide and importin alpha can be measured using biophysical methods like Surface Plasmon Resonance (SPR). For an SPR assay, recombinant importin alpha protein is immobilized onto a sensor chip surface (e.g., a CM5 chip) using amine coupling chemistry. The NLS peptide is dissolved in running buffer (e.g., 10 mM HEPES, 150 mM NaCl, 0.005% Tween-20, pH 7.4). Serial dilutions of the NLS peptide (ranging from 0.1 to 100 microM) are injected over the immobilized importin alpha surface at a constant flow rate (e.g., 30 microL/min). The association phase is measured for 1-2 minutes, followed by a dissociation phase for 2-5 minutes. The sensor surface is regenerated between cycles with a short pulse of low pH buffer (e.g., 10 mM glycine, pH 2.0). The resulting sensorgrams are fit globally to a 1:1 binding model to calculate the association rate (ka), dissociation rate (kd), and the equilibrium dissociation constant (KD). The NLS typically binds importin alpha with affinity in the low nanomolar range.
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| Cell Assay |
A standard cell-based assay for NLS function is the digitonin-permeabilized cell assay. HeLa or other adherent cells are grown on coverslips to ~70% confluency. The cells are washed with ice-cold transport buffer (20 mM HEPES, pH 7.3, 110 mM KOAc, 2 mM Mg(OAc)2, 2 mM DTT). The plasma membrane is selectively permeabilized by incubating the cells on ice for 5 minutes in transport buffer containing digitonin (e.g., 40 microg/mL). After permeabilization, the cells are washed and then incubated with an import reaction mixture. This mixture contains: a fluorescently labeled cargo protein (e.g., BSA-NLS-FITC at 1 microM), recombinant importin alpha/beta (1 microM each), an energy regenerating system (ATP, creatine phosphate, creatine kinase), and the NLS peptide (PKKKRKV) under investigation at various concentrations (0-100 microM). The cells are incubated at 30degC for 30 minutes to allow import to occur. The reaction is stopped by washing with ice-cold transport buffer, and the cells are fixed with 4% paraformaldehyde. The coverslips are mounted on slides with DAPI-containing mounting media. Nuclear fluorescence intensity is then quantified using fluorescence microscopy, and the data are expressed as a ratio of nuclear to cytoplasmic fluorescence to determine import efficiency.
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| Animal Protocol |
An in vivo model for studying NLS-mediated delivery is the use of a xenograft mouse model for imaging. Athymic nude mice (6-8 weeks) are inoculated subcutaneously with 5×10⁶ luciferase-expressing cancer cells (e.g., HeLa-Luc). Once tumors reach approximately 150 mm3, the mice are randomly assigned to treatment groups. A therapeutic nanoparticle, plasmid DNA, or siRNA is conjugated to or formulated with the NLS peptide (PKKKRKV). The conjugate is administered intravenously (e.g., 10-50 mg/kg of NLS-peptide equivalent) once every 2-3 days for 2 weeks. Tumor growth is monitored bi-weekly using calipers. For imaging, the NLS can be conjugated to a near-infrared (NIR) dye like Cy5.5 and injected (10 mg/kg). In vivo fluorescence imaging is performed at 0, 1, 2, 4, 8, 12, and 24 hours post-injection using an IVIS spectrum. At the end of the experiment, tumors and major organs are excised for ex vivo imaging and biodistribution analysis to determine the efficacy of the NLS-mediated nuclear delivery.
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| ADME/Pharmacokinetics |
NLS (PKKKRKV) hydrochloride is a lyophilized powder. It should be stored at -20degC in a sealed container, away from moisture, where it is stable for up to 3 years. For use in experiments, it is readily soluble in water or PBS at a concentration of at least 10 mg/mL. The molecular weight is approximately 919.6 g/mol. The hydrochloride (HCl) salt form is used to improve its solubility and long-term stability. Because it is a small peptide (MW < 1 kDa), it is rapidly cleared from the circulation via renal filtration, leading to a very short plasma half-life (minutes) in vivo. This limits its utility as a standalone therapeutic but makes it an ideal transient delivery agent for research applications.
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| Toxicity/Toxicokinetics |
This product is intended for research use only and is not for clinical use. No specific toxicity data is available for the NLS peptide alone. The peptide itself is derived from a viral protein and is generally considered to be non-toxic and non-immunogenic in most research applications. However, when conjugated to other molecules, the toxicity of the conjugate will depend primarily on the nature of the cargo. The hydrochloride salt form is safe for laboratory use. Standard chemical safety precautions, including the use of lab coats and gloves, should be followed when handling the compound.
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| References |
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| Additional Infomation |
The SV40 NLS (PKKKRKV) is a classic and one of the most powerful and well-characterized NLS sequences known. It is a monopartite NLS, meaning its signal is contained within a single cluster of basic amino acids. This peptide has been used for decades to study the fundamentals of nuclear transport. In modern research, it is an essential tool for developing more effective gene delivery vectors, as the inability of DNA to cross the nuclear envelope is a major barrier to non-viral gene therapy. The hydrochloride salt form is a common synthetic counterion used to improve the physicochemical properties of peptides for research applications. It can be incorporated into lipid nanoparticles (LNPs) or conjugated directly to proteins to improve their delivery to the nucleus.
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| Molecular Formula |
C40H79CLN14O8
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| Related CAS # |
NLS (PKKKRKV);95088-49-6;NLS (PKKKRKV) (TFA)
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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 :~500 mg/mL (~543.71 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 100 mg/mL (108.74 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
(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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