Cell-signaling assays
Assays for detecting the presence and activity of cell-signaling components. These assays include luminescence polarization assays for detecting cell-signaling nucleotides and modulators of receptors and enzymes related to the generation and activity of such nucleotides.
1. A method of testing a compound for modulation of a receptor or enzyme involved in generation of a cyclic nucleotide, the method comprising:
contacting a sample with a tracer comprising a cyclic nucleotide coupled to a luminophore, and with an opposite member of a specific binding pair to the cyclic nucleotide;
illuminating the sample with polarized light, wherein the light is capable of inducing emission of polarized light from the luminophore;
measuring the extent of polarization of light emitted from the luminophore in the presence and in the absence of a compound; and
identifying the compound as a modulator of the receptor or enzyme if the extent of polarization changes in the presence relative to the absence of the compound.
2. The method of claim 1 , wherein the step of identifying includes a step of identifying the compound as an inhibitor of the receptor or enzyme if the extent of polarization increases in the presence relative to the absence of the compound.
3. The method of claim 1 , wherein the step of identifying includes a step of identifying the compound as an activator of the receptor or enzyme if the extent of polarization decreases in the presence relative to the absence of the compound.
4. The method of claim 1 , wherein the step of measuring the extent of polarization of light comprises a step of evaluating a function selected from the group consisting of polarization and anisotropy.
5. The method of claim 1 , wherein the opposite member of a specific binding pair is an antibody.
6. The method of claim 1 , wherein the nucleotide is cyclic AMP (cAMP) or cyclic GMP (cGMP).
7. The method of claim 6 , wherein the nucleotide is cyclic AMP (cAMP).
8. The method of claim 6 , wherein the nucleotide is cyclic GMP (cGMP).
9. The method of claim 1 , wherein the step of contacting includes a step of contacting whole cells or a cell lysate with the compound.
10. The method of claim 1 , wherein the step of identifying includes a step of identifying the compound as an inhibitor or activator of a G-protein-coupled receptor.
11. The method of claim 1 , wherein the step of identifying includes a step of identifying the compound as an inhibitor or activator of adenylyl cyclase.
12. The method of claim 1 , wherein the step of identifying includes a step of identifying the compound as an inhibitor or activator of guanylyl cyclase.
13. The method of claim 1 , further comprising a step of providing the tracer in a lysis buffer.