SapphireTM Insect Transfec-
tion Kit
I nsect cells and lytic baculoviruses
provide a proven method for high-
level expression of full-length mam-
malian proteins. Autographa californi-
ca nuclear polyhedrosis virus (AcNPV)
is used to infect cultured insect cells
(e.g. Spodoptera frugiperda). Expres-
sion of the highly abundant polyhedrin
gene is nonessential in tissue culture
and its strong promoter can be used
for the transcription of foreign genes.
The polyhedrin promoter is maximally
expressed very late stage in infection
when the lytic virus kills the host cells,
resulting in high levels of expression
even for certain toxic proteins. In ad-
dition, many post-translational modifi-
cations similar to those in mammalian
cells are made in insect cells and pro-
teins unable to be expressed in E. coli
have been successfully expressed
in the insect cell system. The Sap-
phire™ Insect Transfection Kit is ideal
for easy and efficient transfection of
DNAs into insect cells. It is optimized
for use with Sapphire™ Baculovirus
DNA and various transfer vectors.
Protocol
Transfection Using the Sap-
phire™ Transfection Kit
1. Seed 2 x 106 Sf9 (Cat. No. ABP-
CEL-10001) or Sf21 (Cat. No. ABP-
CEL-10002) cells onto each 60mm
tissue culture plate. Allow cells to
attach firmly which takes usually 5 to
10 min.
Note: Prepare three plates for each
co-transfection: one for the positive
control, one for the negative control,
and one for the recombinant plasmid
of interest.
2. Remove medium and replace it
with 1 ml of sterile Transfection Buffer
A.
3. Mix 0.5 μg of Sapphire™ or other
baculoviral DNA and 2μg of recom-
binant baculovirus transfer vector
containing gene of interest in a sterile
microfuge tube.
4. Let mixture from Step 3 sit for 1
min at room temperature before add-
ing 1 ml of sterile Transfection Buffer
B.
Allele Biotech-Introducing Cost Effectiveness to Research
5. Mix well and add the mixture from
Step 4 dropwise to cells from Step 2.
For negative control, use Sapphire™ F or Research Use Only. Not for
Diagnostic or Therapeutic Use.
DNA alone without any vector DNA. Purchase does not include or carry
any right to resell or transfer this
For positive control, use pVL1392- product either as a stand-alone
XylE (Cat. No. ABP-BVP-10001) in product or as a component of another
combination with Sapphire™ DNA. product. Any use of this product other
6. Incubate the plates at 27°C for than the permitted use without the
4 hours. After that time, remove medi- express written authorization of Allele
um and replace it with fresh TNM-FH Biotech is strictly prohibited
medium containing 10% fetal bovine
serum (Cat. No. ABP-MED-10001).
7. After 5 days, collect the superna- Website: www.allelebiotech.com
tant of all three plates and infect fresh Call: 1-800-991-RNAi/858-587-6645
cells to amplify the virus. Optionally, (Pacific Time: 9:00AM~5:00PM)
lyse the transfected cells and check Email: oligo@allelebiotech.com
for expression of your protein of
interest.
Box 1 | Product Summary
Catalogue Number ABP-BVD-10003
Component 1 bottle of Transfection Buffer A, 25ml
1 bottle of Transfection Buffer B, 25ml
Storage Store at +4°C.
Stability The transfection buffers are stable for six months
when stored properly.
Box 2 | Related Products
Product Catalogue No.
Sapphiretm Baculovirus DNA (5µg) ABP-BVD-10001
Sapphire™ Baculovirus DNA and Transfection ABP-BVD-10002
Kit
Sf9 Cells (in culture, 107 cells) ABP-CEL-10002
Sf9 Cells (frozen, 10 cells)
7
ABP-CEL-10006
Sf21 Cells (in culture, 107 cells) ABP-CEL-10003
Sf21 Cells (frozen, 10 cells)
7
ABP-CEL-10007
T.ni Cells (in culture, 10 cells)
7
ABP-CEL-10005
T.ni Cells (frozen, 107 cells) ABP-CEL-10008
TNM-FH Insect Culture Medium ABP-MED-10001
Serum Free Insect Culture Medium ABP-MED-10002
Serum Free, Met Free Insect Culture Medium ABP-MED-10003
Grace’s Insect Culture Medium ABP-MED-10004
pVL-1392-XyIE Control Vector ABP-BVP-10001
Insect Lysis Buffer 5X (10ml) ABP-BUF-10010