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Chariot™* is a revolutionary delivery reagent that quickly and efficiently transports biologically active proteins, peptides and antibodies directly into cells. The typical delivery efficiency is 60-95%. Less than two hours after delivery, live cells can be assayed to determine the effects of the introduced material, without the need for fixing. This makes Chariot an ideal tool for functional studies, including delivering inhibitory proteins, labeling organelles, screening peptide libraries and studying protein half-lives & transient complementation.
* Chariot is covered under U.S. Patent No. 6,841,535. Purchase includes the right to use for basic research purposes only. Other use licenses are available, please contact Active Motif Technical Service for additional information.
Chariot™ advantages
- Works in a variety of cell lines (see table on the User Data tab)
- Facilitates functional studies in living cells — no fixing required
- Works in vivo1
- Fast and efficient — 60-95% transfection in less than 2 hours
- No need for expression of fusion proteins
- Non-cytotoxic, serum independent
Why use Chariot™?
Current transfection techniques include cationic liposomes2, calcium phosphate coprecipitation3, electroporation4, microinjection5 and viral vectors6. These methods are useful but are very time-consuming as once transfection is completed, it takes 12-80 hours to detect expression of the gene of interest. A greater drawback is that the cytotoxicity of these methods can make it difficult to determine if any observed changes are caused by the recombinant protein or induced by the transfection method itself.
Chariot is a novel transfection method that eliminates many of the problems encountered with more commonly used procedures. The Chariot peptide forms a non-covalent bond with the macromolecule of interest. This stabilizes the protein, protecting it from degradation and preserving its natural characteristics during the transfection process7, 8. After delivery, the complex dissociates, leaving the macromolecule biologically active and free to proceed to its target organelle. Delivery occurs in the presence or absence of serum and is independent of the endosomal pathway, which can modify macromolecules during internalization. This, coupled with the fact that Chariot is non-cytotoxic, ensures that observed changes are the result of your recombinant protein, and not the transfection method.
Diagram 1: Flow chart of the Chariot process.
Proven performance
Chariot has been used to efficiently transfect a variety of cell lines. As a general rule, the larger the protein to be delivered, the slower it will be delivered; increasing protein size may also reduce delivery efficiency, though increasing delivery time will usually allow for adequate delivery. In contrast, Chariot cannot deliver very small peptides because they do not form a viable complex with the Chariot peptide. Therefore, we recommend that peptides be a minimum of 10 amino acids in length.
Chariot™ delivers for you
Antibody
The ability of Chariot to deliver antibodies was demonstrated using FITC-labeled Actin and Lamp-1 antibodies. These were targeted to Actin filaments (Figure 1) and endosomes (Figure 2), respectively.
Figure 1: Delivery of fluorescent Actin antibody.
Actin filament labeling of human fibroblast (HS-68) cells using a 1/1000 dilution of FITC-labeled Actin antibody. Fixed (A) and unfixed (B) cells were observed two hours post-transfection.
Figure 2: Delivery of fluorescent Lamp-1 antibody.
Endosome labeling of HS-68 cells using a 1/500 dilution of FITC-labeled Lamp 1 antibody. Unfixed cells were observed two hours post-transfection.
Protein
β-galactosidase is a protein that is composed of four identical subunits. The ability of Chariot to deliver protein was demonstrated using a 119 kDa subunit of β-galactosidase (Figure 3).
Figure 3: Delivery of β-galactosidase into HeLa cells.
1 µg of a 119 kDa subunit of β-galactosidase was complexed with Chariot™ Protein Delivery Reagent for 30 minutes and delivered into HeLa cells. Two hours post-delivery, cells were fixed and stained using the β-Galactosidase Staining Kit.
Targeted delivery
The non-covalent Chariot-macromolecule complex dissolves after delivery, leaving the macromolecule biologically active and free to proceed to its target organelle, if it contains specific signaling sequences. This was demonstrated using a 51-mer nuclear protein (Figure 4) and a 32-mer cytoplasmic peptide (Figure 5).
Figure 4: Nuclear-targeted protein delivery.
Chariot was complexed with a fluorescently labeled 51-mer nuclear protein at a 20:1 ratio and delivered into HS-68 cells. Unfixed cells were observed 90 minutes post-delivery.
Figure 5: Cytoplasmic peptide delivery.
Chariot was complexed with a fluorescently labeled 32-mer cytoplasmic peptide at a 20:1 ratio and delivered into mouse 3T3 fibroblasts. Unfixed cells were observed 90 minutes post-delivery.
Chariot speeds and simplifies a variety of functional studies because it efficiently delivers biologically active proteins, peptides and antibodies directly into live mammalian cells. This means you’ll get conclusive data immediately, rather than having to wait days for ambiguous expression data. For additional information, please download the Chariot manual and product profile.
References
- Aoshiba, K. et al. (2003) Am. J. Resp. Cell & Mol. Biol. 28: 555-562.
- Felgner, P.L. et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7417.
- Graham, F.L. and van der Eb, A.J. (1973) Virology 52: 456-467.
- Neumann, E. et al. (1982) EMBO J. 7: 841-845.
- Capecchi, M.R. (1980) Cell 22: 479-488.
- Cepko, C.L. et al. (1984) Cell 37: 1053-1062.
- Morris, M. et al. (1999) J. Biol. Chem. 274 (35): 24941-24946.
- Morris, M. et al. (2001) Nature Biotech 19: 1173-1176.
