CHO-K1 cells were cultured in F12 medium with 10% FBS and seeded at a density of 30,000 cells/well in Isoplate-96 plates (PerkinElmer). The WT (HA-Flag-3GSA-GIPR(22-466)) or mutant GIPR were transiently transfected using Lipofectamine 2000 transfection reagent. The mutant construct was modified by single-point mutation in the setting of the WT construct. Twenty-four hours after transfection, cells were washed twice, and incubated with blocking buffer (F12 supplemented with 33 mM HEPES and 0.1% BSA, pH 7.4) for 2 hr at 37°C. For homogeneous binding, cells were incubated in binding buffer with a constant concentration of 125I-GIP (40 pM, PerkinElmer) and increasing concentrations of unlabeled GIP1-42 (3.57 pM–1 μM) at RT for 3 hr. Following incubation, cells were washed three times with ice-cold PBS and lysed by addition of 50 μL lysis buffer (PBS supplemented with 20 mM Tris–HCl, 1% Triton X-100, pH 7.4). Fifty microliters of scintillation cocktail (OptiPhase SuperMix, PerkinElmer) was added, and the plates were subsequently counted for radioactivity (counts per minute, CPM) in a scintillation counter (MicroBeta2 Plate Counter, PerkinElmer).
125i gip
Manufactured by PerkinElmer
125I-GIP is a radioactive labeling reagent used for the detection and quantification of gastric inhibitory polypeptide (GIP) in biological samples. It provides a direct measurement of GIP levels using radioimmunoassay or similar techniques.
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2 protocols using 125i gip
1
GIPR Receptor Binding Assay in CHO-K1 Cells
2
GIPR Binding Assay in CHO-K1 Cells
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CHO-K1 cells were seeded at a density of 30,000 cells/well in Isoplate-96 plates (PerkinElmer). The WT GIPR or SVs were transiently transfected using Lipofectamine 2000 transfection reagent. Twenty-four hours after transfection, cells were washed twice, and incubated with blocking buffer (F12 supplemented with 33 mM HEPES and 0.1% BSA, pH 7.4) for 2 h at 37°C. For homogeneous binding, cells were incubated in binding buffer with a constant concentration of 125 I-GIP (40 pM, PerkinElmer) and increasing concentrations of unlabeled GIP1-42 (3.57 pM to 1 μM) at RT for 3 h.
Following incubation, cells were washed three times with ice-cold PBS and lysed by addition of 50 μL lysis buffer (PBS supplemented with 20 mM Tris-HCl, 1% Triton X-100, pH 7.4). Fifty µL of scintillation cocktail (OptiPhase SuperMix, PerkinElmer) was added and the plates were subsequently counted for radioactivity (counts per minute, CPM) in a scintillation counter (MicroBeta2 Plate Counter, PerkinElmer).
Following incubation, cells were washed three times with ice-cold PBS and lysed by addition of 50 μL lysis buffer (PBS supplemented with 20 mM Tris-HCl, 1% Triton X-100, pH 7.4). Fifty µL of scintillation cocktail (OptiPhase SuperMix, PerkinElmer) was added and the plates were subsequently counted for radioactivity (counts per minute, CPM) in a scintillation counter (MicroBeta2 Plate Counter, PerkinElmer).
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