Allele Biotech-Introducing Cost Effectiveness to Research
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 modi
-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
Reagent
is
for easy and efcient transfection of
DNAs into insect cells. It is optimized for use with Sapphire™ Baculovirus DNA and various transfer vectors.
Transfection Using the Sap-phire™ Transfection
Reagent
1.
Seed 1 x 10
6
Sf9 (Cat. No. ABP-CEL-10001) or Sf21 (Cat. No. ABP-CEL-10002) cells onto each 35mm tissue culture plate. Allow cells to
attach rmly 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 TNMFH minus serum.
3.
Mix 0.1 μg of Sapphire™ baculo
-
viral DNA and 1μg of recombinant
baculovirus transfer vector containing gene of interest in a sterile microfuge tube containing 100 ul of TNMFH minus serum. In a separate tube pre-
pare transfection mix containing 3 μl
of Insect shuttle transfection reagent
and 97 μl of TNMFH minus serum.
Combine both tubes by pipetting the content of tube containing transfec-tion mix slowly into one containing DNA.
4.
Let mixture sit for 10-15 min at on ice and bring the volume to 1 ml total by adding TNMFH minus serum.
5.
Remove media from 35 mm dish and add the combined mixture from Step 4 dropwise to cells from Step 2. For negative control, use Sapphire™ DNA alone without any vector DNA. For positive control, use pVL1392-XylE (Cat. No. ABP-BVP-10001) in combination with Sapphire™ DNA.
6.
Incubate the plates at 27°C for 4-5 hours. After that time, remove medium and replace it with 2 ml of fresh TNM-FH medium containing 10% fetal bovine serum (Cat. No. ABP-MED-10001).
7.
After 5 days, collect the superna-tant of all three plates and infect fresh cells to amplify the virus. Optionally, lyse the transfected cells and check for expression of your protein of interest.
Sapphire
TM
Insect Transfection
Reagent
Protocol
Box 1 | Product Summary
Catalogue Number
ABP-BVD-10003
Component
75 μl of DNA insect shuttle transfection reagents
(25 reactions)
Storage
Store at +4°C.
Stability
The transfection buffers are stable for six months when stored properly.
F
or Research Use Only. Not for Diagnostic or Therapeutic Use.
Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express written authorization of Allele Biotech is strictly prohibited
Box 2 | Related Products
ProductCatalogue No.
Sapphire
tm
Baculovirus DNA (10rxn)ABP-BVD-10001Sapphire™ Baculovirus DNA and Transfection
Reagent
ABP-BVD-10002Sf9 Cells (frozen, 10
7
cells)ABP-CEL-10006Sf21 Cells (frozen, 10
7
cells)ABP-CEL-10007T.ni Cells (frozen, 10
7
cells)ABP-CEL-10008TNM-FH Insect Culture MediumABP-MED-10001Serum Free Insect Culture MediumABP-MED-10002Grace’s Insect Culture MediumABP-MED-10004pVL-1392-XyIE Control VectorABP-BVP-10001Insect Lysis Buffer 5X (10ml)ABP-BUF-10010
Website: www.allelebiotech.comCall: 1-800-991-RNAi/858-587-6645
(Pacifc Time: 9:00AM~5:00PM)
Email: oligo@allelebiotech.com
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