For non-radioactive detection of receptor/ligand interaction.
This is the second generation of AP-TAG technology. A secreted ligand or soluble receptor can be fused with secreted alkaline
phosphatase (AP) at either its N- or C-terminus to produce an "AP-body". The resulting AP fusion protein can be expressed as a secreted protein
and used directly as highly sensitive affinity agents much like an antibody.
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NOTE: pAPtag-2 and pAPtag-4 plasmid vectors can only be transformed or propagated in E. coli host cells
with P3 episome such as GH2/P3 Supercompetent cells (Cat. No. T601).
The L(left)- and R(right)-AP primers flanking the cloning sites of pAPtag-2 are used in PCR to check for the presence and size of
DNA insert cloned into the vector and for sequence verification of the Ligand-AP or soluble receptor-AP fusion constructs. The L-AP4 and R-AP4 primers (not included in kit) flank the cloning sites of pAPtag-4 and can be purchased separately (see below).
This kit is shipped on dry ice via overnight delivery.
A detailed step-by-step protocol is included.
References:
- Flanagan, J. G. and Leder, P. (1990). The kit Ligand: A cell surface molecule altered in steel mutant fibroblasts. Cell 63, 185-194.
- Cheng, H.J., and Flanagan, J.G. (1994). Identification and cloning of ELF-1, a developmentaly 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. (2000). Alkaline phosphatase fusions of ligands or receptors as in situ probes for staining of cells, tissues and embryos. Methods in Enzymology 327, 17-35.
- Flanagan, J.G., and Cheng, H.-J. (2000). Alkaline phosphatase fusion proteins for molecular characterization and cloning of ligands and receptors. Methods in Enzymology 327, 198-210.
- US patents 5,554,499 and 5,801,000.
See AP-TAG Citation List for more.
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