Purchase of an AP-TAG Ligand-Receptor Interaction Detection System (covered by US patents 5,554,499 and
5,801,000) comes with a limited, single-user and non-transferable sublicense for use in research applications
only. By breaking the seal of the AP-TAG kit , the user agrees to the following terms and conditions:
- No part of the AP-TAG kit, pAPtag plasmid vectors, AP-body plasmid vectors, or their derivatives
(i.e. AP-TAG fusion proteins, etc.) shall be disseminated, propagated, or distributed outside the user's
own laboratory without written permission from GenHunter, which holds world-wide exclusive rights to
the AP-TAG technology in the field of research applications.
- This sub-license does not cover the rights for drug screening or other commercial applications,
which require a separate license. We define "drug screening" as assays that specifically fall under
the scope of our US patent 5,554,499; in particular, the Claim 32 which reads as follows:
"A method for determining the ability of a molecule to interfere with the binding of a ligand of
a known polypeptide receptor to said polypeptide receptor comprising contacting said ligand with said
molecule and with a hybrid molecule comprising said polypeptide receptor covalently bonded to SEAP,
allowing an affinity complex between said hybrid molecule and said ligand to form, and measuring said
affinity complex using a molecule which binds SEAP, the measurement being an inverse measure of said
ability".
Examples of "drug screening" using AP-TAG technology:
- To determine the ability (or IC50) of a drug or drug candidate, such as a therapeutic antibody,
soluble receptor-Fc fusion protein, or small molecular weight inhibitor, to inhibit the interaction
between a ligand and its receptor (or soluble receptor) linked to SEAP (AP). The readout will be a
decrease in AP activity reflecting the extent of inhibition of a soluble receptor-AP bound to its
ligand.
- To conduct high-throughput screening of a compound library for molecules that have the ability
to inhibit the interaction between a ligand and its receptor (or soluble receptor) linked to SEAP
(AP). The readout will be a decrease in AP activity reflecting the extent of inhibition of soluble
receptor bound to the ligand.
If you plan to use AP-TAG technology to conduct the above type of drug screening or other commercial
applications, a separate sublicense is required. Please contact GenHunter at 800-311-8260 or 615-833-0665
for details regarding the terms and conditions or any other questions.
The essence of this invention is to allow a cDNA sequence encoding any secreted polypeptide ligand or
extracellular domain of a receptor to be in-frame fused to human placental secreted alkaline phosphatase
(AP) in pAPtag cloning vectors. The resulting ligand-AP fusion protein, designated AP-body, when
expressed in 293 T Cells, can be secreted at high levels into the culture medium and thus easily
detected by either the AP activity assay or Western blot analysis using antibody against AP. The
Ligand-AP or soluble receptor-AP fusion proteins thus can serve as affinity agents much like antibodies,
which allow the most convenient, safe, and sensitive detection and cloning of their corresponding cell
surface receptors or ligands (ref.1, 2, 3, 4). Unlike the conventional radioactive 125I labeling method,
AP-TAG is safe and does not require ligand/soluble receptor purification.
Since its invention, many important cell surface receptors and ligands have been cloned by AP-TAG technology
including receptors for leptin (ref. 3), Semaphorin III (Ref. 4) and ligands for Kit,
Mek4 and Sek receptor tyrosine kinases (ref. 1, 2).
GenHunter is extremely pleased to be able to add this innovative method into our product line as a powerful
tool for applications downstream of differential display (DD). If you are working with a secreted protein
or cell surface molecule cloned by DD or other methods, AP-TAG technology may allow you to
functionally characterize these genes further.
Comparison of AP-TAG technology and the conventional radioactive 125I ligand-labeling method:
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| | AP-TAG | 125I labeling |
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| Ligand purification | Not required | Required |
| Labeling Reaction | Not required | Required |
| Hazardous | No | Yes |
| Detection | Colorimetric | Scintillation counting |
| Sensitivity | High | High |
| Cell Staining | Yes | No |
| Expression Cloning | Yes | Yes |
| Ligand-Receptor Binding Kinetics/Affinity | Yes | Yes |
Schematic illustration of AP-TAG technology and
its major applications
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AP fusion construct
Create an in-frame fusion of your cDNA encoding a secreted ligand or soluble receptor with either the N- or C-terminus of secreted alkaline phosphatase (AP) in pAPtag expression vectors.
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AP fusion protein expression
After transfecting the above AP fusion plasmid construct into 293T or NIH 3T3 cells, the expression of the secreted AP fusion protein (AP-body) can be measured by either colorimetric AP activity assay or immunoblotting (or IP) with antibody to AP.
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Receptor/ligand binding assay
The culture medium containing the secreted AP fusion protein can be used directly to measure the presence or absence of a cell
surface receptor (or ligand) of interest by assaying the AP activity bound to the cells. The secreted AP alone is used as a negative control.
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in situ staining of receptor/ligand
The secreted AP fusion protein can be used much like an antibody to detect the tissue distribution of a cell surface receptor/ligand of interest. An expression cDNA library thus can be made with mRNA isolated from tissues that express the highest level of the receptor/ligand for subsequent expression cloning.
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Receptor cloning of receptor/ligand
The secreted AP fusion protein can be used as a probe to clone a cell surface receptor or ligand of interest by traditional expression cloning strategy (panning).
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