Biology Reference
In-Depth Information
4. Binding buffer: 50 mM Tris-Cl pH 7.4, 10 mM MgCl 2 , 100 mM NaCl
5. Bovine serum albumin (BSA, Sigma-Aldrich)
6. Wash buffer: 50 mM Tris-Cl pH 7.4 (pH must be adjusted within a temperature
between 4 and 10 C)
7. Protease inhibitors: phenanthroline, pepstatin, leupeptin, and aprotinin (Sigma-
Aldrich)
8. Radioligand at levels sufficient to occupy 20% of receptors, [ 3 H]-PGF2 a
(Perkin Elmer, 150-240 Ci/mmol)
9. GTP g S (Sigma)
10. SARSTEDT tubes for binding: 5 ml, 75
12 mm, PS (cat. #55.476), and caps
(cat. #65.809)
11. Fisher glass fiber circles (2.4 cm, cat. #09-804-24C)
12. 50 ml PLASTIBRAND pipette tip (cat. #Z332798) and repeater pipette
13. Millipore 1225 sampling manifold or equivalent (cat. #xx2702550)
14. Scintillation tubes and scintillation liquid for tritiated ligands
15.
b -scintillation counter for quantification of [ 3 H]-ligand binding
9.2 METHODS
9.2.1 Cell culture and transfection
1. Day 1: HEK 293 cells (or other cell types such as vascular smooth muscle cells)
are cultured in MEM or DMEM containing L -glutamine, 10% FBS, and
antibiotics, at a density of 500,000 cells (quantity may vary depending on
transfection method) per 10 cm dish. Cell lines are grown at 37 Cin5%CO 2 .
a. To verify the effects of the G protein or an allosteric ligand on radioligand
binding of the receptor of interest, only one condition, with the receptor
expressed alone, needs to be considered.
b. To verify the effects of a receptor partner on radioligand binding of the
receptor of interest, two conditions are necessary: the receptor of interest
expressed alone or expressed with the receptor partner.
2. Day 2: cells are transfected with the desired receptor(s) (1.5-2 m g), using the
calcium phosphate method (other transfection methods can be used). Use an
empty vector to assure similar DNA quantity in each transfection condition.
Alternatively, cells stably expressing receptor(s) of interest can be used.
3. Day 3: change media.
4. Day 4: perform binding experiment (see Section 9.2.2 ).
9.2.2 Radioligand binding and dissociation kinetics
1. At room temperature, rinse the plates with 5 ml PBS.
2. To detach the cells, add 2 ml of PBS-2 mM EDTA per plate and wait for 5 min.
3. Gently scrape and collect the cells. Put the cells on ice.
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