just wondering, why is it important to add the homogenate to the test tube last in a competitive radioligand binding assay?
No need to bother about the addition in receptor binding assay.when you add everything in the reaction mixture like Hot, cold, receptor then only the reaction will start.So it does,t matter.
Can anyone help me with a protocol for the radioligand whole cell binding assay? something like 3H Estradiol binding in mouse/human ER beta transfected HeLa cells?
first Prepare membrane as follows, then try protocol given below that
Membrane Preparation Buffer: (pH 6.8)
25 mM 2,2-[hydroxymethyl]ethyl amino ethanesulfonic acid (TES)
0.5 mM EDTA
0.2 mM MgCl2
1 mM 1,10-Phenanthroline
Membrane Storage Buffer:
Membrane preparation buffer supplied with
Membrane preparation protocol:
Washed cells twice with ice-cold PBS
Pelleted by centrifugation at 2000g for 10 min
Cells were resuspended in membrane preparation buffer (pH 6.8)
Homogenized on ice at 20,500 rpm for 10 min
Membrane were isolated by centrifugation at 45,000g for 30 min at 4°C
Pelletes were resuspended in Membrane storage buffer
Membrane concentration was estimated using Bradford’s method
Binding Assay: Membrane was mixed with binding buffer (50mM Tris, 10 mM MgCl2, 1mM EGTA, 0.1% BSA, complete protease inhibitor cocktail, pH7.4) at a concentration of 40μg/well. The antagonist plates were prepared with a final concentration of 10μM in DMSO. 50μl of both membrane solution and radioligand ( 3H Nonspecific binding for the -------- receptors was determined. The plate was incubated for two hours at room temperature, incubations was terminated by rapid filtration through UniFilter-96 plates GF/B, presoaked for at least 2 h in polyethylenimine 0.3%, and using a MicroMate 96 Cell Harvester. Plates and filters were then washed 5 times with 0.5 ml aliquots of Tris buffer (50 mM, pH 7.4, 4 °C). Filters were dried and soaked in Microscint 40 (50 μl/well, Perkin Elmer), and bound radioactivity was counted by a Top Count Microplate Scintillation Counter (Perkin Elmer). Each experiment (n) was performed in triplicate (association and saturation studies) or in duplicate (inhibition experiments).Competition data were analyzed using Graph Pad Prism software. Inhibitor dissociation constants (Ki) were calculated from IC50 values using the relationship described by Cheng and Prusoff (1973).