Biosep sec s2000 column

The HEK-293 cells transfected with FSHR and a coupled G protein were purchased from Merck Millipore (Milford, MA, USA). The GS HiRange kit was purchased from PerkinElmer Corp (Waltham, MA, USA). The Waters Xevo G2-XS Q-TOF mass spectrometer, equipped with an Acquity UPLC system, Acquity glycan BEH amide UPLC column and HPLC Alliance 2690 were purchased from Waters (Milford, MA, USA). The BIOSEP SEC-S2000 column was purchased from Tosoh Bioscience GmbH (Stuttgart, Germany). Amicon Ultra 3K centrifugal filters were purchased from Merck Millipore (Watford, UK). Water used in experiments was purified with a Milli-Q system from Merck Millipore (Milford, MA, USA). GlycoClean S cartridges were purchased from Prozyme Inc. (Hayward, CA, USA). The cell counter was purchased from Chemometec (Allerod, Denmark) and the homogeneous time-resolved fluorescence (HTRF) plate reader was purchased from Agilent (Santa Clara, CA, USA).

The protein content of each sample was determined using size-exclusion–high-performance liquid chromatography (SE–HPLC). To determine content, the r-hFSH samples were diluted in Kolliphor 188 buffer and analyzed by HPLC Alliance 2690 (Waters, Milford, MA, USA) using a BIOSEP SEC-S2000 column (Tosoh Bioscience GmbH, Stuttgart, Germany) at room temperature. Elution was carried out using a buffer containing 0.1 M sodium phosphate and 0.1 M sodium sulfate at pH 6.7, at a flow rate of 1 mL/min. Detection was carried out at 214 nm and protein content was determined by calibration against the RHS.