Biology Reference
In-Depth Information
4. Dose protein content of whole cells using 35-50 m l of the PBS-EDTA-
resuspended cells.
5. Pellet the cells by centrifugation (500
g ) and resuspend the cells in binding
buffer containing 0.2% BSA and protease inhibitors to have a concentration of
2 m g/ m l (final quantity of cells needed will be 100 m g, may vary depending
on the cell type and transfection efficiency and can be predeterminated
for optimal binding). Alternatively, membranes (especially for the effect of the
activated G proteins on radioligand binding to the receptor) can be purified
from the cells by homogenizing them with a Teflon potter (by doing 20
strokes on ice), followed by a 30 min centrifugation at 40,000
g at 4 C
and used for this experiment, instead of whole cells. The quantity of membrane
to use for optimal binding has to be predetermined.
6. Prepare the radioligands: Use enough radioligand to occupy no more than
20% of the receptors at equilibrium in a final volume of 100 m l binding
buffer. This represents
50,000-100,000 cpm of [ 3 H]-PGF2 a at
specific activity of 150-240 Ci/mmol, which has an affinity for FP in the
4-6 nM range. To calculate nonspecific binding, cells are incubated with
the same quantity of radioligand and an excess (1000
the K i ) of cold ligand
(see next step).
7. Prepare the binding reaction in the 5 ml SARSTEDT tubes (in duplicate or
triplicate), for different times (e.g., 0, 1, 2, 5, 15, and 30 min. In this example,
the “0” time point is the total binding), and the nonspecific binding control. Add
in the following order, at room temperature (for a final volume of 400 m l):
a. 220 m l of binding buffer
b. 50 m l of cold 8
ligand (for nonspecific binding) or buffer (for total binding)
c. 50 m l of vehicle (control) or cold 8
allosteric ligand (to assess the effect
of this ligand on the orthosteric radioligand binding to the receptor of
interest) or 80 m M GTP g S (to have 10 m M final concentration, to assess
the effect of the activated G protein(s) on the orthosteric radioligand binding
to the receptor of interest)
d. 80 m l of radioligand
e. 50 m l of cells (100 m g protein) to start the binding reaction
f. Leave the reaction 1 h to reach binding equilibrium (or longer if necessary,
to assess the time of binding equilibrium, association kinetics can also be
assessed)
8. During this time, equilibrate glass fiber circles in binding buffer containing
0.2% BSA (w/v).
9. After radioligand binding to the receptor has reach equilibrium, start the
dissociation kinetics experiment by adding 50 m l of cold orthosteric 10
ligand,
starting with the latest time point (in this example, 30 min) to the shortest.
10. Before the end of the time course experiment, prepare the manifold of the cell
harvester for the filtration of the binding reaction by putting the equilibrated
glass fiber circles on the manifold and rinse them once with 2 ml of cold
50 mM Tris-HCl, pH 7.4.
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