For transfection with pAPtag vectors
293T is a human embryonic kidney cell line commonly used for transfection assays. Due to the expression of the large T antigen in the cell, plasmids with SV40 origin of replication (such as pAPtag-2, pAPtag-4, and pAPtag-5) can be transiently transfected and give extremely high levels of expression of AP fusion proteins (e.g. ligand-AP fusion proteins). Thus, we strongly recommend using this cell line for your production of AP fusion proteins with pAPtag vectors. The fusion
proteins can be easily monitored 2-3 days after transfection by alkaline phosphatase assay (See our AP Assay Reagent A, Cat. No. Q501) or by Western blot using AP Antibody. But for long term
production of AP fusion proteins, we recommend that a stable cell line be cloned by co-transfecting with a puromycin or hygromycin-resistant
plasmid (293T is G418 resistant).
Detailed protocol included.
| | Cat. No. | Size |
|---|
| 293 T Cells | Q401 | 5 x 106 Cells/Vial |
|
For production of high levels of AP alone
The 293T/pAPtag-4 stable cell line is used to produce high levels of secreted human placental alkaline
phosphatase (AP) which can be used as a negative control for a ligand-AP or soluble receptor-AP fusion protein in cell surface binding assays or cell staining. High level production of secreted AP can be achieved with sub-confluent to confluent culture a few days after medium change. The secretion of AP can be monitored easily with the culture medium by AP activity assay using GenHunter AP Assay Reagent A (Cat. No. Q501).
Detailed protocol included.
| | Cat. No. | Size |
|---|
| 293T/pAPtag-4 Stable Cell Line | Q402 | 5 x 106 Cells/Vial |
|
For use as a selectable marker for transfection of cultured mammalian cells
The pSV-Hygro vector confers hygromycin resistance and the pBabe-Puro vector confers puromycin resistance when co-transfected into cells.
| |
Selectable Marker |
Cat. No. |
Size |
|---|
| pSV-Hygro co-transfection vector |
Hygromycin |
Q455 |
10 µg |
| pBabe-Puro co-transfection vector |
Puromycin |
Q456 |
10 µg |
|
For construction of expression cDNA libraries
This cloning vector has been used extensively to construct mammalian expression cDNA libraries (see below references).
The vector contains an SV40 origin of replication and the major adeno late promoter (PMAL) in front of Neo resistance cDNA insert
flanked by an EcoR I and Xho I site. Using the Stratagene cDNA Synthesis Kits generally results in cDNA ends with EcoR I and Xho I
sites, which can be directionally cloned into the pMT21-NeoR vector.
| | Cat. No. | Size |
|---|
| pMT21-Neo Mammalian Expression Cloning Vector | Q454 | 20 µg |
|
For expression cloning of cell surface receptor/ligand using AP fusion proteins (AP-bodies)
This kit consists of a cos-1 host cell line (commonly used for expression cloning by panning) and a positive
control receptor/ligand-AP pair. An expression cDNA library potentially containing the receptor/ligand gene of interest can be
transiently transfected into these cells. Positive cDNA pools can be identified by staining the transfected cells with your AP
fusion protein. Cells over-expressing the corresponding cell surface receptor/ligand will be stained blue with GenHunter AP Activity
Assay Reagent S (see figure below).
The Kit ligand positive control vector [containing a 1 kb cDNA encoding the transmembrane form of Kit ligand (stem cell factor),
Ref. # 4] can be transfected into the cos-1 host cell line. Cells overexpressing cell surface Kit ligand will be stained blue by
soluble receptor Kit-AP fusion protein using GenHunter AP Assay Reagent S.
References:
- Cheng, H.J., and Flanagan, J.G. (1994). Identification and cloning of ELF-1, a developmentally expressed ligand for Mek4 and Sek receptor tyrosine kinases. Cell 79, 157-168.
- Tartaglia, L.A. et al. (1995). Identification and expression cloning of a leptin receptor, OB-R. Cell 83, 1263-1271.
- He, Z. and Tessier-Lavigne, M. (1997). Neuropilin is a receptor for the axonal chemorepellent Semaphorin III. Cell 90, 739-751.
- Flanagan, J.G. et al. (1991). Transmembrane Form of the kit Ligand Growth Factor is Determined by Alternative Splicing and is missing in the SId Mutant. Cell 64, 1025-1035.
|
Expression cloning of cell surface receptor/ligand by panning
This kit is shipped on dry ice via overnight delivery. A detailed step-by-step protocol is included. |
| | Cat. No. | Size |
|---|
| Expression Cloning Kit | Q450 | 20 µg |
|
Individual components for the Expression Cloning Kit sold separately
|
| Description | Cat. No. | Volume |
| cos-1 Host Cell Line | Q451 | 1x106 cells/vial |
| Kit-AP fusion protein (media) 1 unit/mL | Q452 | 10 mL |
| Kit-AP ligand (stem cell factor) positive control vector | Q453 | 100 µg |
|
