Cell-free assay product and method of use thereof for measuring activity of frizzled receptors
The present invention relates to cell free assays for measuring receptor activity, especially for measuring a constitutive or a non-constitutive activity of frizzled re-ceptors and uses thereof. The present invention further concerns a method for measuring a constitutive or non-constitutive activity of a frizzled receptor and a method for obtaining an active frizzled receptor ligand.
1. A cell-free assay product for measuring a constitutive activity of frizzled receptors comprising at least one G protein, at least one system for measuring G protein activity and at least one membrane protein with at least one frizzled receptor, wherein a concentration of the G protein is approximately 0.4 μg/ml or less.
2. The product of claim 1 , wherein a concentration of the G protein is between approximately 0.002 μg/ml and 0.3 μg/ml.
3. The product of claim 2 , wherein a concentration of the G protein is between approximately 0.005 μg/ml and 0.15 μg/ml.
4. The product of claim 3 , wherein a concentration of the G protein is approximately 0.1 μg/ml in relation to approximately 4 μg/ml to 10 μg/ml of the membrane protein.
5. The assay product of claim 1 wherein the system for measuring G protein activity is selected from the group consisting of fluorescent, radioactive and spectrophotometric techniques.
6. The assay product of claim 1 further comprising at least one frizzled receptor ligand.
7. The assay product of claim 6 , wherein the frizzled receptor ligand is at least one modulator of frizzled receptor activity.
8. The assay product of claim 6 , wherein the frizzled receptor ligand is at least one antagonist.
9. The assay product of claim 6 , wherein the frizzled receptor ligand is at least one agonist, comprising inverse agonists.
10. The assay product of claim 6 , wherein the frizzled receptor ligand is at least one natural ligand.
11. The assay product of claim 6 , wherein the frizzled receptor ligand is at least one artificial ligand, comprising recombinant ligands.
12. The assay product of claim 6 wherein the frizzled receptor ligand and/or frizzled receptor is/are expressed by a vector.
13. The product of claim 12 , wherein the vector is expressed in bacteria or in Escherichia coli.
14. A cell-free assay product for measuring a non-constitutive activity of frizzled receptors comprising a G protein, at least one system for measuring G protein activity and at least one membrane protein with at least one frizzled receptor, wherein a concentration of the G protein is approximately 0.3 μg/ml or higher.
15. The product of claim 14 , wherein a concentration of the G protein is between approximately 0.035 μg/ml and 10 μg/ml.
16. The product of claim 15 , wherein a concentration of the G protein is approximately 0.4 μg/ml.
17. The product of claim 16 , wherein a concentration of the G protein is approximately 0.5 μg/ml in relation to approximately 4 μg/ml to 10 μg/ml of the membrane protein.
18. A method comprising screening frizzled receptor ligands, measuring levels of frizzled receptor ligands, measuring levels of frizzled receptors, or obtaining active frizzled receptor ligands using the assay product of claim 1 .