We obtained MacQ’s absorption spectra by placing the rhodopsin under the cytomegalovirus (CMV) promoter and transfecting HEK cells at a total volume of 200 mL for three days. We added all-trans retinal (5 µM) and cultured the cells in the dark for 3 h. We pelleted the cells, lysed them using a sonicator in 50 mM Tris with 2 mM MgCl2 at pH 7.3, and pelleted again. Finally, we homogenized the lysate in a solution (pH 7.2) containing 30 mM K2HPO4, 20 mM KH2PO4, 300 mM NaCl, and 1.5% N-Octyl-β-D-Glucopyranoside (Sigma) and pelleted once more. We obtained the absorption spectrum using the supernatant and a spectrophotometer (Tecan Safire 2 UV-Vis).
Safire 2 uv vis
Manufactured by Tecan
The Safire 2 UV-Vis is a versatile spectrophotometer designed for absorbance measurements in the ultraviolet and visible light spectrum. It features a xenon flash lamp as the light source and a monochromator to select the desired wavelengths. The instrument is capable of performing various photometric techniques, including absorbance, transmittance, and fluorescence measurements.
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2 protocols using safire 2 uv vis
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Rhodopsin Absorption Spectrum Measurement
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Rhodopsin Absorption Spectrum Measurement
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We obtained MacQ’s absorption spectra by placing the rhodopsin under the cytomegalovirus (CMV) promoter and transfecting HEK cells at a total volume of 200 mL for three days. We added all-trans retinal (5 µM) and cultured the cells in the dark for 3 h. We pelleted the cells, lysed them using a sonicator in 50 mM Tris with 2 mM MgCl2 at pH 7.3, and pelleted again. Finally, we homogenized the lysate in a solution (pH 7.2) containing 30 mM K2HPO4, 20 mM KH2PO4, 300 mM NaCl, and 1.5% N-Octyl-β-D-Glucopyranoside (Sigma) and pelleted once more. We obtained the absorption spectrum using the supernatant and a spectrophotometer (Tecan Safire 2 UV-Vis).
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