1 kw halogen lamp

The cDNA of lamprey parietopsin was tagged with the monoclonal antibody Rho 1D4 epitope sequence (ETSQVAPA). The tagged parietopsin cDNAs were inserted into a pcDNA3.1 (Invitrogen). Pigment expression in HEK293S cells and purification were carried out as described previously [26 (link)]. Briefly, to reconstitute the pigment, the expressed proteins were incubated overnight with excess 11-cis retinal or 11-cis 3-dehydororetinal. The pigments were then extracted using 1% dodecyl β-D-maltoside (DM) in 50 mM HEPES buffer (pH 6.5) containing 140 mM NaCl (buffer A). To purify the pigment, pigments in the crude extract were bound to 1D4-agarose, washed with 0.02% DM in buffer A (buffer B), and eluted with buffer B containing the 1D4 peptide. The spectra of purified parietopsin reconstituted with 11-cis retinal and 11-cis 3-dehydroretinal were recorded with 100 mM hydroxyl amine. The pigment absorption spectra were recorded at 0°C with a Shimadzu UV2450 spectrophotometer. Green light was generated using a 1-kW halogen lamp (Philips) equipped with a 550-nm interference filter, and an O53 glass cutoff filter (Toshiba), respectively.

The opsin expression and purification were performed as described previously [34 (link), 35 (link)]. Briefly, the opsin expression vectors were transfected into HEK293S cells or COS-1 cells (SV40-transformed African green monkey kidney cell line) using the polyethylenimine (PEI) transfection method. The transfected cells were harvested two days after the transfection. After addition of 11-cis retinal, opsin-based pigments were extracted with 1% dodecyl β-D-maltoside (DM) in HEPES buffer (pH 6.5) containing 140 mM NaCl and 3 mM MgCl₂ (buffer A), bound to 1D4-agarose, washed with 0.02% DM in buffer A and eluted with buffer A containing 0.02% DM and C-terminal peptide of bovine rhodopsin as described.
The absorption spectra of the opsin-based pigments were recorded at 4°C by using a Shimadzu UV2450 spectrophotometer. Blue lights were supplied by a 1-kW halogen lamp (Philips) with a 470 nm interference filter (MZ0470; Asahi Spectra Co., Ltd., Tokyo, Japan).