Several biochemical and biophysical methods are available to determine ligand binding affinities between a biological target and its ligands, most of which require purification, labelling or surface immobilisation. These measurements, however, remain challenging in regards to membrane proteins, as purification processes require their extraction from their native lipid environment, which may in turn impact receptor conformation and functionality. In this study, we have developed a novel experimental procedure using microscale thermophoresis (MST) directly from cell membrane fragments, to determine different ligand binding affinities to a membrane protein, the dopamine D2 receptor (D2R). In order to achieve this, two main challenges had to be overcome: determining the concentration of dopamine D2R in the crude sample; finding ways to minimize or account for non-specific binding of the ligand to cell fragments. Using MST, we were able to determine the D2R concentration in cell membrane fragments to approximately 36.8 ± 2.6 pmol/mg. Next, the doses-responses curves allowed for the determination of KD, to approximately 5.3 ± 1.7 nM, which is very close to the reported value. Important details of the experimental procedure have been detailed in this paper to allow the application of this novel method to various membrane proteins.< Leer menos