Transport™ Protein Delivery Reagent
Instructions for Use
Introduction
The Transport Protein Delivery Reagent is a noncytotoxic, newly discovered peptide (patent pending) that
translocates itself as well as attached cargo molecules
into cultured cells when added to the culture medium.
Cargo molecules can be proteins, peptides,
oligonucleotides or small molecules which contain a free
thiol group (i.e.,cysteine).
•
Efficient delivery of cargo molecules– up to 100% of
the cultured primary cells or cell lines.
•
Non-cytotoxic peptide.
Preparation of Transport™ Reagent-Cargo
Complex Solution
Using Table 1, calculate the final volume of the Transport
Reagent-cargo complex solution needed to perform your
experiment.
Table 1.
Plate/Dish Type
Complex Volume (ml)
96 well
0.05–0.1 per well
24 well
12 well
0.3–0.6 per well
0.6–1.2 per well
6 well
35 mm
1.5–3.0 per well
1.5–3.0 per plate
•
Non-invasive delivery.
The conjugation of the Transport Reagent and a cargo
molecule occurs through the 2-pyridyldithio group on the
reagent that forms a disulfide bond with any free cysteine
situated on the cargo molecule.
Receiving Instructions
60 mm
3.0–6.5 per plate
100 mm
10.0–17.0 per plate
Upon receipt, store at -20°C.
Kit Contents
1. 50566 Transport Protein Delivery Reagent 400 µg
2. 50567 beta-galactosidase 50 µl
Precautions
THESE PRODUCTS ARE FOR RESEARCH USE ONLY. Not
approved for human or veterinary use, or for application
to humans or animals, or for use in vitro diagnostic or
clinical procedures.
NOTE: This substance is a peptide designed to transport
molecules across cell membranes. While it is not known
to be hazardous, the effects of other molecules carried
into cells may have undesired or detrimental effects.
Avoid direct contact.
Instructions
Reconstitution of Transport™ Reagent
1.
2.
Reconstitute 1 vial of lyophilized Transport Reagent
in 279 µl of sterile water. As a result, a 500 µM stock
is generated.
Aliquot the reconstituted reagent into microº
centrifuge tubes. Store aliquots at –20 C for up to 6
months or at –80ºC for up to 1 year. Avoid
repeating freeze-thaw cycles.
Cambrex Bio Science Rockland, Inc.
www.cambrex.com/bioproducts
biotechserv@cambrex.com
Orders: 800-638-8174 Technical Service: 800-521-0390
Copyright © 2005 Cambrex Bio Science Rockland, Inc.
To Prepare 5 ml of Transport™ Reagent-Cargo
Complex Solution
1.
2.
Thaw aliquots of Transport Protein Delivery Reagent
and the cargo protein at room temperature for
immediate use.
Add required volume of cargo protein to a 15 ml tube.
Use Table 2 to generate the desired final
concentration of the cargo molecule.
Table 2.
Amount of cargo protein to use for a final concentration of
100 nM cargo protein in 5 ml solution
Encoding
DNA
Amino
Acids
kDa
µg of protein in 5 ml
of 100 nM solution
90 bp
810 bp
2.7 kb
30
270
900
3
30
100
1.7
15
50
NOTE: Protein solution should not contain any reducing
agents, such as DTT, ß-Mercaptoethanol or glutathione.
3.
4.
5.
Add 10 µl of Transport Protein Delivery Reagent to
the cargo molecule. Final volume should be
approximately 100 µl. Adjust volume when necessary
with sterile distilled water.
Incubate for 15-30 minutes at room temperature.
Dilute the prepared complex with 3.0-5.0 ml complete
cell culture media (containing serum or defined
proteins).
Addition of Transport™ Reagent-Cargo
Complex to Cells
Testing for Transduction Activity with
ß-galactosidase (beta-gal).
1.
Transduction activity of the Transport Reagent can be
easily assessed by use of beta-gal as a control cargo
protein.
1. One day prior to the protein delivery experiment seed
50,000 cells per well in a 24 well plate. Cells should
be about 50% confluent on the day of the
experiment.
2.
3.
4.
Seed appropriate number of cells per well one day
prior to protein delivery experiment.
On the day of the experiment, aspirate media and
add the appropriate volume of Transport ReagentCargo complex solution to each well (see Table 1 for
volumes).
Incubate the plate for 30 minutes to 2 hours in
standard growth conditions (37ºC, 5% CO2).
Wash the cells twice with PBS before performing the
final analysis.
2.
3.
4.
Ordering Information
Catalog No.
Description
50568
Transport Protein Delivery Reagent
For more information contact Technical Service at
(800) 521-0390 or visit our website at
www.cambrex.com.
5.
6.
7.
8.
To 80 µl of water in a 15 ml tube, add 10 µl of the
reconstituted Transport Reagent and 10 µl of the
beta-gal control solution (included in kit), mix, and
incubate for 15 minutes at room temperature.
Add 5ml of complete culture media to the complex.
Prepare additional 1:2 and 1:4 dilutions if necessary.
Wash cells once with fresh media. Add 0.6 ml of the
Transport Reagent-beta-gal complex solution to the
wells.
Incubate for 90 minutes in standard growth
conditions (i.e., 37ºC, 5% CO2 in a humidified
incubator).
Wash the cells 3 times with PBS and fix the cells with
a solution of 2% Formaldehyde and 0.2%
Glutaraldehyde in PBS for 10 minutes at room
temperature.
Remove the fixing solution and wash twice with PBS.
Stain for beta-gal activity using standard betagalactosidase staining protocols or kits (i.e., Roche
cat# 1828673). Analyze cells under a light
microscope.
NOTE: The conditions presented above represent the
starting guidelines. To obtain the best performance of the
product, we recommend optimization of the Transport
Protein complex concentration, as well as the cell culture
density.
Cambrex Bio Science Rockland, Inc.
191 Thomaston Street
Rockland Maine 04841
To Order:
Phone: (800) 638-8174
Fax: (800) 362-5552
E-mail: biocustserv@cambrex.com
Outside USA:
Phone: (207) 594-3400
Fax: (207) 594-3491
Technical Service:
Phone: (800) 521-0390
Fax: (800) 362-1133
E-mail: biotechserv@cambrex.com
Homepage: www.cambrex.com
Cambrex Bio Science Copenhagen, ApS.
Risingevej 1
DK-2665 Vallensbaek Strand
Denmark
Phone: 45 43 56 74 00
Fax: 45 43 56 74 03
Cambrex Bio Science Wokingham, Ltd.
1 Ashville Way
Wokingham, Berkshire RG41 2PL
United Kingdom
Phone: 44 118 979 5234
Fax: 44 118 979 5231
For Research Use Only.
Transport is a trademark of Cambrex or its subsidiaries.
©2005 Cambrex Bio Science Rockland, Inc. All rights
reserved.
18289-0405-05
Cambrex Bio Science Rockland, Inc.
www.cambrex.com/bioproducts
biotechserv@cambrex.com
Orders: 800-638-8174 Technical Service: 800-521-0390
Copyright © 2005 Cambrex Bio Science Rockland, Inc.