Hana3A cells were used for recombinant OR expression to test specificity of the OR antibodies. The Hana3A cells were kindly provided by H. Matsunami (Duke University Medical Center, Durham, NC). The Hana3A cells were maintained under standard conditions as previously described [76 (link)] and grown on cover slips in 24-well plates. Cells were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol with 300 ng of OR plasmid and 60 ng of mRTP1S plasmid (constructed using standard PCR methods).
For the antibody specificity studies, the OR6B2 coding sequence was PCR amplified from human genomic DNA using specific primer pairs (for: 5’-GCATATGAATTCATGAGTGGGGAGAATGTCACC-3’ and rev: 5’-GCATATGCGGCCGCTCAGTGTGAAGTTTGACCCAAGC-3’) that amplify the complete open reading frame and contain the EcoRI and NotI restriction sites for further subcloning into the pCI plasmid (Promega, Madison, USA), which contain the coding sequence for the N-terminal rhodopsin tag (rho-tag, first 20 amino acids of rhodopsin). All plasmid constructs and PCR products were verified by Sanger sequencing.
For the antibody specificity studies, the OR6B2 coding sequence was PCR amplified from human genomic DNA using specific primer pairs (for: 5’-GCATATGAATTCATGAGTGGGGAGAATGTCACC-3’ and rev: 5’-GCATATGCGGCCGCTCAGTGTGAAGTTTGACCCAAGC-3’) that amplify the complete open reading frame and contain the EcoRI and NotI restriction sites for further subcloning into the pCI plasmid (Promega, Madison, USA), which contain the coding sequence for the N-terminal rhodopsin tag (rho-tag, first 20 amino acids of rhodopsin). All plasmid constructs and PCR products were verified by Sanger sequencing.
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