Enabling Epigenetics ResearchChariot™
Catalog No. 30025 & 30100(Version C3)

Copyright 2023 Active Motif, Inc.

Revision

Date

Description of Change

C2July, 2023

The “Sterile H20” is now described as “Water”. However, thecomposition has not changed.

C3

October, 2023

The Active Motif beta-galactosidase Staining Kit is no longer available.
Prepare reagents for staining as described in the manual or use any
commercially available beta-galactosidase staining kit such as theInvitrogen beta-Gal Staining Kit (catalog no. K146501).

Information in this manual is subject to change without notice and does not constitute a
commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any
warranty of any kind, either explicit or implied. Information may be changed or updated inthis manual at any time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated,
in whole or in part, without the prior written consent of Active Motif, Inc. This documentation
is proprietary information and protected by the copyright laws of the United States andinternational treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2023 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All
rights reserved.
All trademarks, trade names, service marks or logos referenced herein belong to theirrespective companies.
For research use only. Not for use in diagnostic procedures.

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Contents
Overview  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Kit Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Quality Control  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  2
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
Protocols  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Preparation of Chariot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Preparation of β-galactosidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Transfection Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  5
Transfection Protocol for Suspension Cells  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  7β-galactosidase Staining Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  8
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

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Overview
Chariot™ is a patented* transfection reagent suitable for the delivery of proteins, peptides andantibodies into a variety of cultured mammalian cells. Chariot Kits come in two sizes and canbe used with many different plate sizes as indicated in the table below.
Plate

Chariot™

Transfections per Kit(25-reaction Kit)

Transfections per Kit(100-reaction Kit)

96-well

0.5 μL

300

1200

24-well

2 μL

75

300

12-well

3.5 μL

42

171

6-well or 35 mm

6 μL

25

100

60 mm

20 μL

7

30

100 mm

50 μL

3

12

Kit Contents
Component

25 rxns

100 rxns

Composition

Storage

Chariot Transfection Reagent

0.3 mg

1.2 mg

PBS

1 mL

1 mL

β-galactosidase (Positive Control)

25 µg

25 µg

-20°C for up
to 6 months

Sterile H2O

1 mL

1 mL

-20°C

-20°C for up
to 6 months
1.5 mM KH2PO4
150 mM NaCl2
5 mM Na2HPO4-20°C

Quality Control• Chariot Transfection Reagent is functionally tested by delivery of protein and antibody
into many different cell lines (including HS-68, NIH 3T3, and HeLa). Please visit our
website for a complete list of cell lines that have been tested.
• Chariot Transfection Reagent is tested for the absence of bacterial and fungal
contamination in DMEM cell culture media supplemented with 10% FBS.
• Chariot Transfection Reagent is non-cytotoxic at the recommended concentrations.

*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 Services for additional information.

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Introduction
Chariot™ is a revolutionary new transfection reagent capable of efficiently delivering proteins,
peptides and antibodies into cultured mammalian cells in less than two hours.
Current transfection techniques include microinjection1, calcium phosphate coprecipitation2,
cationic liposomes3, viral vectors4 and electroporation5. These methods are capable oftransporting DNA into cells, but the techniques can be cumbersome and even cytotoxic.
Once transfection has been completed, the researcher must wait 12-80 hours posttransfection to detect expression of the gene of interest.
Recently, novel methods to deliver proteins have been reported and commercialized, i.e.
HIV-1 TAT, Drosophila Antennapedia homeotic transcription factor and herpes simplex virus1 DNA binding protein VP22 (for a recent review, see ref. 6). Transduction via TAT-fusion
proteins results in inactivation and denaturation of the protein. To deliver an active protein,correct renaturation is required upon internalization. In addition, TAT must be covalently
linked to the compound or macromolecule to be delivered by a chemical reaction.
Penetratin 1 is a 16 amino acid peptide corresponding to the third helix of the homeodomainof Antennapedia protein. Activated Penetratin has an N-terminal pyridyl disulfide that is used
to covalently couple it to the macromolecule to be delivered. However, chemical coupling
can be cumbersome and requires the macromolecule being delivered to carry a freethiol group.
Another delivery system utilizes the translocation properties of the 38 kDa herpes simplex
virus-1 DNA binding protein VP22. VP22 must be fused to the peptide/protein to be delivered
and, therefore, requires the construction of a suitable expression vector.
Chariot is a 2843 dalton peptide and unlike these current transfection techniques, Chariot
forms a non-covalent complex with the protein, peptide or antibody of interest. This
completely bypasses the transcription-translation process associated with gene expression,
reducing the time until the cells can be assayed from days to under two hours. The
Chariot-macromolecule complex stabilizes the macromolecule and helps to protect it fromdegradation during the transfection process7, 8. Upon internalization, the complex dissociates
and the macromolecule is free to proceed to its target organelle. Moreover, efficient delivery
is observed at 4ºC, suggesting that the delivery mechanism is independent of the endosomal
pathway8. Therefore, the macromolecule is not subjected to the conditions of that pathway,
which can modify the structure of the macromolecule during internalization. Chariot is noncytotoxic and serum-independent8. It is ideal for in vivo studies because fixing is not required.

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Flow Chart of Processactivemotif.com|

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Protocols
Preparation of Chariot
Chariot is provided as a lyophilized powder and arrives at room temperature. Store at -20ºC
for long term storage.
25-reaction Kit: 0.3 mg of Chariot is provided. Resuspend the entire contents of the Chariot
vial in 150 µL sterile H2O. Mix gently by tapping tube.
100-reaction Kit: 1.2 mg of Chariot is provided. Resuspend the entire contents of the Chariot
vial in 600 µL sterile H2O. Mix gently by tapping tube.
After resuspension, store at -20°C in small aliquots to avoid repeated freeze/thaw cycles.
Note:

If you will be transfecting a peptide or low molecular weight protein (< 10 kDa),
you must make an additional 1:10 dilution of the resuspended Chariot
solution in sterile H2O before use. This will improve transfection efficiency by
preventing the formation of aggregates.

Note:

Diluting Chariot in DMSO has been shown to improve delivery of some
antibodies. It has also provided more uniform delivery of some proteins.
Please refer to the sections in the Troubleshooting Guide (page 10) for
more information.

Preparation of β-galactosidase
β-galactosidase is an enzyme that hydrolyses β-galactosides, such as lactose and theartificial chromogen X-gal. β-galactosidase is composed of four identical subunits. Whenβ-galactosidase hydrolyses X-gal, it produces a blue color that can be visualized under abright field microscope. A 119 kDa subunit of the β-galactosidase protein is provided in thiskit as a positive control for the Chariot Transfection Reagent.
The β-galactosidase positive control protein is provided as a lyophilized powder. Store at-20ºC. 25 µg of protein is provided. Resuspend the entire contents of the β-galactosidase
positive control vial in 100 µL sterile H2O. This makes a 0.25 µg/µL stock. Use 1.0 µg
β-galactosidase per transfection reaction. After resuspension, aliquot and store at -20°C toavoid repeated freeze/thaw cycles.
When the β-galactosidase positive control protein is transfected, the cells must be stained toassay for the efficiency of the transfection. A detailed staining protocol and buffer recipes areprovided on page 8. Alternatively a commercially available beta-galactosidase Staining Kit
such as the Invitrogen beta-Gal Staining Kit (catalog no: K146501) can be used.

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Transfection Protocol
These conditions are recommended as guidelines only. Efficient transfection may require
optimization of reagent concentration, cell number and exposure time of cells to theChariot-macromolecule complex. Conditions should also be optimized for each cell line andkept consistent to obtain reproducible results. This procedure has been optimized for thetransfection of adherent cells using the β-galactosidase positive control. A protocol for using
suspension cells can be found on page 7.
1.

Seed cells according to the following table:
Plate

2.

Surface Area

Cells

Growth Medium

96-well

20 mm2

1.0 x 104

0.2 mL

24-well

200 mm2

0.05 x 106

0.5-1.0 mL

12-well

401 mm0.1 x 10

6-well or 35 mm

6

1-2 mL

962 mm2

0.3 x 106

3-5 mL

60 mm

2827 mm2

0.8 x 10

65 mL

100 mm7854 mm2

2.2 x 106

10 mL

Incubate the cells at 37ºC in a humidified atmosphere containing 5% CO2 until thecells are 40-50% confluent.

Note:
3.

2

The transfection efficiency may be sensitive to culture confluency, so it may
be necessary to optimize cell density for each cell line.

Preparation of diluted macromolecule: Dilute your protein, antibody or peptide
of interest in the appropriate volume of PBS as indicated in the table below. For
antibodies or proteins, we recommend using 0.5-1 µg per transfection reaction.
For peptides or proteins that are < 10 kDa, we recommend using 100-500 ng pertransfection reaction.

Note:

The amount of macromolecule used must be optimized, we recommend
performing a titration of the protein/antibody or peptide quantities.

Plate

Final Volume of Macromolecule
Dilution in PBS

96-well (or 8-well chamber slide)

10 µL

24-well

50 µL

12-well

100 µL

6-well or 35 mm

100 µL

60 mm

200 µL

100 mm200 µL* If you are using 96-well plates, you may wish to reduce the amount of protein or antibody
used to 0.2 µg and amount of peptide to 50 ng.

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4.

Preparation of diluted Chariot: In a separate tube, dilute the appropriate volume ofChariot into sterile water as indicated in the table below. For low molecular weightproteins and peptides, a 1/10 dilution of the resuspended Chariot must be madebefore use to ensure optimal transfection.
Sterile H20
for Chariot
DilutionProtein/Antibody*
Volume of Chariot

Peptide/LMW Proteins**
Volume of a 1/10
Dilution of Chariot

96-well

10 µL

0.5 µL0.5 µL

24-well

50 µL

2 µL

2 µL

12-well

100 µL

3.5 µL

3.5 µL

6-well or 35 mm

100 µL

6 µL

6 µL

60 mm

200 µL

20 µL

20 µL

100 mm200 µL

50 µL

50 µL

Plate

For multiple transfections, do not use a master mix that exceeds the volume required for 4
transfections (6-well plate size) per tube, as this may cause aggregation. (Note: This does not
apply to peptide transfections)*Detection of fluorescent antibodies in cells requires a large amount of antibody to bedelivered. It may be necessary to increase the amount of Chariot and antibody used.
**Chariot interacts via hydrophobic interactions. Each peptide or protein will have a differenthydrophobicity, which is more apparent with lower molecular weight molecules. The amountof Chariot may need to be titrated to determine the optimal amount.

5.

Preparation of Chariot-macromolecule complex: Add the diluted macromoleculefrom Step 3 to the diluted Chariot from Step 4 and incubate at room temperature for
30 minutes to allow the Chariot-macromolecule complex to form.

6.

Transfection of cells: Aspirate medium from cells to be transfected and wash cells
once with PBS.

7.

Overlay the cells with the Chariot-macromolecule complex. Next, add the appropriate
amount of serum-free medium to the plate as indicated in the table below. Gently
rock the plate to ensure even delivery.
Volume of ChariotMacromolecule
Complex

Serum-Free
MediumFinal
Transfection
Volume96-well

20 µL

80 µL

100 µL

24-well

100 µL100 µL200 µL

12-well

200 µL150 µL350 µL

6-well or 35 mm

200 µL400 µL

600 µL

60 mm

400 µL

1600 µL

2000 µL

100 mm

400 µL

4600 µL

5000 µL

Plate

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8.
9.

Incubate at 37ºC in a humidified atmosphere containing 5% CO2 for one hour.

Continue to incubate at 37ºC in a humidified atmosphere containing 5% CO2 for 30
minutes to 2 hours.
Optional: If your cells are sensitive to serum starvation you can choose to add
complete growth medium to the cells. Do not remove the Chariot-macromolecule
complex. We suggest adding the minimum amount of complete growth medium
needed as the presence of serum can interfere with Chariot transfection efficiency.
Peptide:0.5-1 hour

Protein:

1-2 hours

Antibody:

2 hours

10. Process the cells for observation or detection assays. Cells may be fixed orobserved directly.

Transfection Protocol for Suspension Cells
These conditions are recommended as guidelines only. Efficient transfection may require
optimization of reagent concentration, cell number and exposure time of cells to the Chariotmacromolecule complex. Conditions should also be optimized for each cell line and kept
consistent to obtain reproducible results.
1.

Seed cells according to the following table:
Plate

Surface Area

Cells

96-well

20 mm2

1.0 x 104

24-well

200 mm2

0.05 x 10

12-well

401 mm2

0.1 x 106

1-2 mL

6-well or 35 mm

962 mm2

0.3 x 106

3-5 mL

60 mm

2827 mm2

0.8 x 1065 mL

100 mm7854 mm

2.2 x 1010 mL

2Growth Medium
0.2 mL
6

6

0.5-1.0 mL

2.

The Chariot-macromolecule complex is assembled in the same manner as describedfor adherent cells. See steps 3-5 on pages 5-6.

3.

Collect the suspension cells by centrifugation at 200-400 x g for 5 minutes. Remove
the supernatant.

4.

Wash the cells twice with 1X PBS.

5.

Centrifuge at 200-400 x g for 5 minutes to pellet the cells. Remove the supernatant.

6.

Resuspend the cell pellet in the Chariot-macromolecule complex. Add serum-free
medium to achieve the Final Transfection Volume. (See step 7 page 6)

7.

Incubate at 37ºC in a humidified atmosphere containing 5% CO2 for one hour.

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8.

Continue to incubate at 37ºC in a humidified atmosphere containing 5% CO2 for 30
minutes to 2 hours.
Optional: If your cells are sensitive to serum starvation you can choose to add
complete growth medium to the cells. Do not remove the Chariot-macromolecule
complex. We suggest adding the minimum amount of complete growth medium
needed as the presence of serum can interfere with Chariot transfection efficiency.

9.

Process the cells for observation or detection assays. Cells may be fixed orobserved directly.

β-galactosidase Staining Protocol
The solutions below can be used to stain and determine the percentage of cells that are
successfully transfected with the beta-galactosidae protein. Alternatively a commerciallyavailable beta-galactosidase Staining Kit such as the Invitrogen beta-Gal Staining Kit (catalog
no: K146501) can be used.
Make Stock Solutions
Solution

Storage

400 mM potassium ferricyanide-20ºC

400 mM potassium ferrocyanide-20ºC

200 mM magnesium chloride-20ºC

20 mg/mL X-gal

5-bromo-4-chloro-3-indolyl-β-D-galactopyranosideresuspended in DMF (N, N-Dimethyl-formamide)

10X PBS-20ºC in the dark
-20ºC

10X Fixing Solution
20% formaldehyde
2% glutaraldehyde
in 10X PBS-20ºC

Staining Protocol
Note:

Use volumes appropriate for the size of the plate or dish. Ensure that the
Fixing and Staining Solutions cover the cells.

1.

Remove the growth medium from the transfected cells and rinse 3 times with 1X PBS.

2.

Add 1X Fixing Solution. (Dilute the 10X stock in sterile water to make a 1X solution.)
Incubate at room temperature for 5-10 minutes.

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3.

Prepare the Staining Solution. Staining Solution should be made fresh each time.
Solution

Final Volume

250 µL 400 mM potassium ferricyanide

4 mM

250 µL 400 mM potassium ferrocyanide

4 mM

250 µL 200 mM magnesium chloride

2 mM

1.25 mL X-gal (20 mg/mL in DMF)

1mg/mL23 mL 1X PBS

23 mL

Total Volume

25 mL

4.

Rinse the cells twice with 1X PBS.

5.

Add the Staining Solution to the cells and incubate at 37ºC for 30 minutes to 2 hours.

6.

Check the cells under a microscope. Calculate the percent of cells transfectedwith β-galactosidase.
Total no. of blue cells
Total no. of cells

x 100 = % transfection

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9References
1.

Capecchi, M.R. (1980) Cell 22: 479-488.

2.

Graham, F.L. and van der Eb, A.J. (1973) Virology 52: 456-467.

3.

Felgner, P.L. et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7417.

4.

Cepko, C.L. et al. (1984) Cell 37: 1053-1062.

5.

Neumann, E. et al. (1982) EMBO J. 7: 841-845.

6.

Schwarze, S.L. et al. (2000) Trends Cell Biol. 10: 290-295.

7.

Morris, M. et al. (1999) J. Biol. Chem. 274 (35): 24941-24946.

8.

Morris, M. et al. (2001) Nature Biotech. 19: 1173-1176

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10Troubleshooting Guide
ProblemPossible Cause

Recommendation

Low Transfection
Efficiency

Cell density

Use adherent cells at a confluency of 40-50%

Chariot or macromolecule
dilutions were performedin larger volumes thanrecommended

Dilute the macromolecule into the final volume of
PBS as recommended. Dilute Chariot in the volumeof sterile water recommended. It is necessary to
make the Chariot macromolecule complex in aconcentrated solution. For multiple transfections,do not use a master mix that exceeds the volume
required for 4 transfections (6-well plate size) per
tube, as this may cause aggregation.

Chariot and macromolecule
were not complexed for 30
minutes

Combine the Chariot dilution and themacromolecule dilution together and allow thecomplex to form at room temperature for
30 minutes.

Improper storage of Chariot

Chariot may form aggregates over time if notstored properly. You may need to sonicate theChariot dilution just before complex formation. We
recommend placing the Chariot dilution in a water
bath and sonicating the water at close proximitywith an ultrasonic processor. At Active Motif, weroutinely use Sonics Vibra-cell™ for 1 minute at an
amplitude of 30 and a 1-second pulse.

Medium that contained
serum was used to dilute
the Chariot-macromolecule
complexRepeat the transfection using serum-free medium
to dilute the Chariot-macromolecule complex to the
Final Transfection Volume.

Exposure of Chariotmacromolecule complex tocells was insufficient

Leave the Chariot-macromolecule complex on thecells for a minimum of two hours.

Using a cell line other thanthose tested with Chariot

Try the transfection in a cell line proven to workwith Chariot. Please visit our website for completein formation on cell lines that have been tested

A suboptimal amount ofmacromolecule was used forthe transfection

You may need to titrate the amount ofmacromolecule (protein, peptide, or antibody) usedin the transfection.

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ProblemPossible Cause

Recommendation

A suboptimal amount ofChariot was used for thetransfection

You may need to titrate the amount of Chariot
used in the transfection. Chariot interacts via
hydrophobic interactions. Each peptide or protein
will have a different hydrophobicity and may requirea different amount of Chariot. Depending on thesensitivity required for antibody detection it may
be necessary to alter the amount of Chariot used.
Detection of fluorescently labeled antibodies incells requires a large amount of antibody to bedelivered. This may mean increasing the amount ofChariot used.

Diluting Chariot into DMSO
may improve antibody
delivery

Diluting Chariot into DMSO has improved thedelivery of some antibodies. If delivery of yourantibody is low, try diluting Chariot into 60-80%
DMSO instead of water. After the Chariot dilution is
combined with the antibody dilution, the complex
mix ture will have a final concentration of 30-40%
DMSO. This may need to be lowered for certain celllines. However, a final concentration of complex
mixture below 10-20% DMSO has not been shown
to improve antibody delivery.

Protein delivery is
localized to a few
regions of the cell

Diluting Chariot into DMSO
may make protein delivery
more uniform

With some proteins that we have tested, deliveryof the protein seems to be localized to specificregions in the cell. Diluting Chariot into DMSO
instead of water has made the delivery of someof these proteins more uniform. Dilute the Chariot
into 40-60% DMSO. After the Chariot dilution is
combined with the protein dilution, the complex
mixture will have a final concentration of
20-30% DMSO.

Aggregate formation

Improper storage of Chariot

Chariot may form aggregates over time if notstored properly. You may need to sonicate theChariot dilution just before complex formation.
We recommend placing the Chariot dilution in awaterbath and sonicating the waterbath with anultrasonic processor, such as a Sonics Vibra-cell™,for 1 minute at an amplitude of 30 and a
1-second pulse.

Excess Chariot used

Decrease the amount of Chariot used

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ProblemPossible Cause

Recommendation

Signs of cytotoxicity
Note: Chariot is noncytotoxic to cells at
the recommended
concentrations

Transfected macromolecule
may be cytotoxic

Transfect the macromolecule at a lower
concentration. Compare cells only, cells with
Chariot alone and cells with macromolecule alone.
Chariot should not affect cell viability. Transfect the
β-galactosidase control protein.

Culture may becontaminated withmycoplasma

Treat the cells to eliminate mycoplasma.

Cells may not be healthy

Check the incubator (CO2 and temperature levels).
Check the media

Too much DMSO present

Reduce the final concentration of DMSO to a final
concentration below 40%

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Technical Services

China

Japan

EuropeNorth America

If you need assistance at any time, please call or send an e-mail to Active Motif Technical
Service at one of the locations listed below.

Toll free:877 222 9543

Direct:

760 431 1263

Fax:

760 431 1351

E-mail:tech_service@activemotif.com

UK Free Phone:

0800/169 31 47

France Free Phone:

0800/90 99 79

Germany Free Phone:

0800/181 99 10

Direct:+32 (0)2 653 0001

Fax:

+32 (0)2 653 0050

E-mail:eurotech@activemotif.com

Direct:+81 (0)3 5225 3638

Fax:

+81 (0)3 5261 8733

E-mail:japantech@activemotif.com

Direct:(86)-21-20926090

Cell Phone:
E-mail:18521362870
techchina@activemotif.com

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