384-well white flat-bottom polystyrene, not-treated microplates, non-sterile (Corning, 3705)
Assay buffer: 25-mM HEPES, pH 7.4, 5-mM MgCl2
, 1-mM CaCl2
, 100-µg/ml bacitracin, and 0.2% (w/v) protease-free BSA.
GraphPad Prism™ software (GraphPad Software).
Human recombinant somatostatin sst4 receptor membrane preparation produced in CHO-K1 cells was used in conjunction with (3-[125I] iodotyrosyl11) Somatostatin-14(tyr11) and PEI PS SPA Imaging Beads. Non-specific binding (NSB) was determined in the presence of 2-µM somatostatin-28. The standard assay format was as follows.
1. Reagents were added in the following order: assay buffer or buffer containing 2% (v/v) DMSO solution, unlabeled ligand (NSB wells), labeled ligand, and premixed bead and membrane. Total assay volume was 40 µl.
2. Diluted membrane and bead were precoupled at room temperature immediately prior to assay addition.
3. Wells contained 10 µl (~ 55 000 dpm) of 1.25-nM (3-[125I] iodotyrosyl11) Somatostatin-14(tyr11) (final concentration 0.313 nM), 62.5 µg of SPA Imaging Bead,
0.313 µg of receptor preparation added together in a 20-µl volume, and 10 µl of assay buffer in the absence of competing ligand. For competition assays, 10 µl of competing ligand prepared in assay buffer containing 2% (v/v) DMSO was added with (3-[125I] iodotyrosyl11) Somatostatin-14(tyr11) (10 µl, as above), precoupled bead, and receptor (20 µl), also giving a total assay volume of 40 µl.
4. NSB wells contained 10 µl of 1.25-nM (3-[125I] iodotyrosyl11) Somatostatin-Page: All 1 2 3 4 5 Related biology technology :1
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