Protein Delivery

Chariot™
simple, efficient
protein delivery

Chariot™ is a revolutionary delivery reagent that efficiently transports
biologically active proteins, peptides and antibodies directly into
cultured mammalian cells in less than two hours.
Chariot advantages
Fast and efficient — up to 95% delivery in under 2 hours
Non-covalent binding preserves the delivered protein’s function
Works in a variety of cell lines, including hard-to-transfect cells
Facilitates functional studies in living cells — no fixing required
Non-cytotoxic, serum independent

Chariot™ – simple, efficient protein delivery
Chariot™ is a revolutionary delivery reagent that efficiently transports biologically active proteins, peptides andantibodies directly into cultured mammalian cells in less than two hours1, 2. The typical delivery efficiency is 65-95%.
After delivery, living cells can be assayed to determine the effects of the introduced materials, without the needfor fixing. This makes Chariot ideal for a wide variety of functional studies, including delivery of inhibitory proteins,organelle labeling, screening peptide libraries and transient complementation studies.
Current transfection techniques often use
microinjection3, calcium phosphate coprecipitation4, cationic liposomes5, viral vectors6
and electroporation7. While these methods
transport DNA into cells, they are often
inconvenient and time-consuming. Aftertransfection, the researcher must wait
overnight to several days to detect geneexpression. A greater drawback is that thecytotoxicity of these methods can make it
difficult to determine if any observedchanges are caused by the recombinant protein or by the transfection method itself.

Chariot™

Chariot is non-cytotoxic and efficiently delivers proteins into a wide range of cell lines,
including hard-to-transfect PC-12 and primarycells. It has proven to be effective on bothadherent and suspension cells. As efficientdelivery occurs at 4°C, the Chariot process
is independent of the endosomal pathway1,
which can modify macromolecules duringinternalization.

Add the Chariot/macromolecule
complex to the cells

Macromolecule
1 hr at 37°C in
5% CO2The Chariot advantage
Chariot is a peptide that forms a non-covalent complex with the macromolecule to bedelivered, which stabilizes the protein, protecting it from degradation during delivery
and preserving its function. Upon addition
to cells, the complex is rapidly internalized,
where it dissociates1. The Chariot peptide
localizes to the cell nucleus where it isdegraded, leaving the macromolecule free toproceed to its target organelle. By directly
delivering protein, peptide or antibody,
Chariot completely bypasses the transcription-translation process associated with gene
expression, saving hours to days of time.

Complex forms in 30 min atroom temperature

Assay formacromolecule activity

Incubate at 37°C for
30 min to 2 hr

Add complete medium (useserum if cells require it)

Diagram 1: Flow chart of the Chariot process.
Chariot is very easy to use. Simply combine it with your macromolecule, wait for 30 minutes, then add to your cells.
Activity can be assayed in as little as 90 minutes post-delivery.

Proven performance
Chariot’s transfection success across multiple
cell lines makes it ideal for any cell system.
It has been used to efficiently deliver proteins, peptides and antibodies directly into
fibroblasts, granulosa cells, protoplasts,
endothelial cells, epithelial cells and neuronalcells. Chariot’s high delivery efficiency ineven difficult-to-transfect cells gives you
flexibility in your cell choice. The process
is also serum-independent, so delivery canoccur in the presence or absence of serum.
Chariot enables you, not your delivery
reagent, to decide which cell lines are best touse in your experiments.

Specific cell lines that have been successfullytransfected using Chariot include:293
CEM-SS
NIH/3T3
JurkatCHO
LLC-PK
Saos-2
NRKHS-68
COS-7
C2C12
PC-12
NCI-H295
NRKIMR90
WI-38

HeLa
MDA-MB-231
Hep G2
CV-1
SIGC
ArabidopsisU87mg
HCA2

www.activemotif.com

Deliver biologically active proteins
The ability of Chariot to deliver biologically
active proteins was demonstrated using
p27kip1, a 27 kDa cyclin-dependent kinase
inhibitor that causes cell-cycle arrest in G1
phase. Flow cytometry (Figures 1A & 1B) andbromodeoxyuridine (BrdU) labeling (Figure 1C)of three different cell lines were performed

following delivery of p27kip1 by Chariot.
HS-68, Jurkat and WI-38 cells were arrested
in G0 phase by serum deprivation for 48
hours, and then released by addition ofserum for three hours. 100 μl of 50 nM p27kip1
protein was complexed with Chariot for 30minutes. Chariot alone, p27kip1 alone and theAA

Chariot-p27kip1 complex were added to thecells. The inability of > 90% of the cells thatreceived the Chariot-p27kip1 complex to progress beyond G1 phase demonstrates efficientdelivery of active p27kip1.

100BBCCCells in G1 (%)

80
60
40
200
G1

101520(h)G20G1101520(h)0Jurkat

Chariot alone

WI-38

HS-68

Chariot-p27kip1 complex

G2Figure 1: Cell cycle arrest by delivered peptide.
Flow cytometry of HS-68 cells was performed 0, 10, 15 and 20 hours after delivery of the samples. Cells that received Chariot alone (A) and p27kip1 alone (data not shown) were able
to progress into G2 phase. Over 90% of the cells that received the Chariot-p27kip1 complex (B) remained in G1 phase. BrdU incorporation experiments were also carried out 14 hours
after delivery of the samples. The cells were labeled with BrdU, fixed and then incubated with anti-BrdU antibody and a fluorescent secondary antibody. The percentage of cells inG1 phase was determined by counting cells on an immunofluorescence microscope (C). Data generously provided by Dr. Gilles Divita, Biophysics Dept., CNRS, Montepellier, France.

Deliver gene silencing reagents
In addition to the original Chariot used todeliver proteins, peptides and antibodies,
Active Motif has developed Chariot II, aunique formulation of Chariot that can beused to deliver our exclusive gripNA™ genesilencing reagent. gripNAs are a modified
form of negatively charged Peptide NucleicAcids, which display superior sequencespecificity compared to conventional genesilencing reagents. In addition, gripNAs areresistant to nuclease degradation. The ability

to deliver gripNAs using Chariot II is a uniqueadvantage as Chariot is non-cytotoxic compared with other lipid-based transfection
reagents. And, as Chariot II cannot be usedwith other nucleic acid based gene silencing
tools such as morpholinos or siRNA, gripNAs
provide you with a better alternative. Visit
the Active Motif gripNA web resource atwww.activemotif.com/gripna for all theup-to-date information you need to design,order and use gripNAs.

North America 877 222 9543 Europe +32 (0)2 653 0001 Japan +81 (0)3 5225 3638

Figure 2: Chariot II delivery of Fluorescein-tagged
gripNA.
An 18-mer gripNA probe (1 μM) labeled on its 3´ endwith Fluorescein was complexed with Chariot II in
PBS and incubated for 30 minutes at 37°C, then overlaid onto cultured HS-68 cells for 1 hour. Cells werewashed extensively prior to observation. Data provided by Dr. L. Chaloin, Dr. M. Morris and Dr. G. Divita,CNRS, Montpellier, France.

www.activemotif.com

Deliver antibodies directly into live cells
Chariot successfully delivers a variety ofantibodies directly into live mammalian cells
using our quick and simple protocol. The
ability of Chariot to deliver antibodies intocells was demonstrated using FITC-labeledanti-Actin and anti-Lamp 1 antibodies. The
labeled antibodies were targeted to actin
filaments (Figure 3) as well as to endosomes(Figure 4), and observed after delivery byfluorescence microscopy.

Figure 3: Delivery of fluorescent Actin antibody.
Actin filament labeling of human fibroblast (HS-68)
cells using a 1/1000 dilution of an anti-Actin antibody.
Unfixed cells were observed 2 hours post-delivery.

Figure 4: Delivery of fluorescent Lamp-1 antibody.
Endosome labeling of HS-68 cells using a 1/500
dilution of anti-Lamp 1 antibody. Unfixed cells
were observed 2 hours post-delivery.

Figure 5: 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 6: Chariot delivery of peptide.
Chariot was complexed with a fluorescently labeled
32-mer cytoplasmic peptide at a 20:1 ratio anddelivered into human fibroblasts. Unfixed cells wereobserved 90 minutes post-delivery.

and antibodies directly into live mammaliancells. This means you’ll get conclusive data
immediately, rather than having to wait days

for ambiguous expression data. To find more
information about Chariot, please visit ourwebsite at www.activemotif.com.

Targeted delivery
An advantage of the non-covalent Chariotmacromolecule complex is that, afterdelivery, the macromolecule is biologically
active and free to proceed to its target
organelle, providing it contains specific signaling sequences. This unique property wasdemonstrated using a 51-mer nuclear protein(Figure 5) and a 32-mer cytoplasmic peptide(Figure 6).

Get Chariot and get results
Chariot speeds and simplifies a variety offunctional studies because it efficiently
delivers biologically active proteins, peptides

Ordering information
Product
Chariot™

Format25 rxns
100 rxns
75 rxnsb-Galactosidase Staining Kit

Catalog No.
30025
30100
35001

CONTENTS & STORAGE

REFERENCES

Sufficient reagents are included for either 25 or 100 reactions,with each reaction being the equivalent of a 35 mm plate or asingle well of a six-well plate. Chariot Kits contain Chariot Protein
Delivery Reagent, b-galactosidase positive control, PBS and sterile
H20. b-Galactosidase Staining Kits contain Glutaraldehyde fixative,
X-gal, MgCl2, potassium ferrocyanide and potassium ferricyanide.
Sufficient reagents are included to perform 75 assays in 35 mmdishes. Store all components at -20°C. All reagents are guaranteedstable for 6 months when stored properly.

1. Morris, M. et al. (2001) Nature Biotech. 19: 1173-1176.
2. Morris, M. et al. (1999) J. Biol. Chem. 274(135): 24941-2494.
3. Capecchi, M.R. (1980) Cell 22: 479-488.
4. Graham, F.L. and van der Eb, A.J. (1973) Virology 52: 456-467.
5. Felgner, P.L. et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7417.
6. Cepko, C.L. et al. (1984) Cell 37: 1035-1062.
7. Neumann, E. et al. (1982) EMBO J. 7: 841-845.

North America 877 222 9543 Europe +32 (0)2 653 0001 Japan +81 (0)3 5225 3638