Hmw calibration kit

Solubilized crude membranes were analyzed by fluorescence size exclusion chromatography (FSEC) on a Superose 6 10/300 column attached to an $Ä$ KTA Purifier (GE Healthcare, USA), using FSEC buffer (20 mM TRIS-HCl, 0.15 M NaCl, 0.03% DDM). 1 μM Astemizole was added to the buffer in experiments involving Astemizole. The effluent from the Superose 6 10/300 column was coupled to a fluorescence detector (Shimadzu Prominence RF-20A), to measure fluorescence and visualize the elution profile of the GFP tagged hERG channel. To estimate the molecular weight of the solubilized hERG-TEV-GFP-His ₈ protein, we used the HMW calibration kit from GE Healthcare dissolved at 20 mg/ml in FSEC buffer. The molecular masses were: Ovalbumin 43 kDa; Conalbumin 75 kDa; Aldolase 158 kDa; Ferritin 440 kDa; Thyroglobulin 669 kDA; Blue Dextran 2000 kDa. The elution volume for Blue Dextran defined the void volume.

30μL crude lysate samples of Alexa488-DDM or DIBMALP-β2AR were run of Yarra 1.8μm SEC-x300 2.5mL column (Phenomenex, CA, US) using shimadzu prominence HPLC system. Running buffer consisted of 20mM HEPEs, 150mM NaCl, 5% glycerol, and 0.03% DDM for DDM- β₂AR sample only. FSEC took place at a flow rate of 0.2mL/min and 0.2mL fractions collected. Samples were excited at 488nm, and emission collected at 520nm. GE HMW calibration kit was use as the standard.

Proteins were loaded onto a Superose™ 6 10/300 column equilibrated in PBS/Az using an ÄKTA FPLC system at 0.3 ml/min and the A₂₈₀ of the eluate monitored continuously. Mass standards were from a commercial HMW calibration kit (GE Healthcare). All buffers and samples were filtered (0.45 µm) before use.

Solubilized crude membranes were analyzed by fluorescence-detection size exclusion chromatography (FSEC) on a Superose 6 Increase 200 10/300 GL column attached to an ÄKTA Purifier (GE Healthcare, USA), using FSEC buffer (20 mM TRIS-HCl, 0.15 M NaCl, 0.03% DDM). The effluent from the column was coupled to a fluorescence detector (Shimadzu Prominence RF-20A) to measure fluorescence and visualize the elution profile of the GFP tagged AQPs. To estimate the molecular weight of the solubilized AQP-TEV-GFPHis₁₀ proteins, we used the HMW calibration kit from GE Healthcare dissolved at 20 mg/ml in FSEC buffer. The molecular masses were: Ovalbumin 43 kDa; Conalbumin 75 kDa; Aldolase 158 kDa; Ferritin 440 kDa; Thyroglobulin 669 kDA; Blue Dextran 2000 kDa. The elution volume for Blue Dextran defined the void volume.

Fluorescence detection size exclusion chromatography (FSEC) analysis of solubilized crude membranes was done on an ÄKTA Purifier (GE Healthcare, USA) using a Superose 6 10/300 column and FSEC buffer (20 mM TRIS–HCl, 0.15 M NaCl, 0.03% DDM pH 7.5). The samples were analyzed with and without addition of CHS 0.026% (w/v) and/or 5 mM KCl. The Superose 6 10/300 column effluent was coupled to a fluorescence detector (Shimadzu Prominence RF-20A). This facilitated fluorescence measurements and visualization of the GFP protein elution profiles. Molecular weight estimation of the solubilized channels was done by a comparison to the HMW calibration kit from GE Healthcare dissolved in FSEC buffer to a concentration of 20 mg/ml. The molecular masses of the kit components were: Blue Dextran 2000 kDa–the elution volume of which was used as definition of void volume; Thyroglobulin 669 kDA; Ferritin 440 kDa; Aldolase 158 kDa; Conalbumin 75 kDa; Ovalbumin 43 kDa.