Protocol detail

Antibody Generation for Visual Proteins

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The antibodies against the lamprey parietopsin and parapinopsin were generated against the C-terminal 55-, 50-amino-acid region of the proteins, respectively. The anti-β-arrestin antibody was generated against 269 amino acids (V29–E297) of the protein. The antigens were prepared using the pMAL Protein Fusion and Purification System (New England Biolabs), following the previously reported methods [10 (link)], and immunized to the chicken (anti-parietopsin antibody), mouse (anti-parapinopsin antibody), and guinea pig (anti-β-arrestin antibody). Antiserums obtained from these immunized animals were used for immunohistochemistry. The anti-VGLUT (Synaptic Systems, #135 503), anti-Go-type G protein (MBL, #551), and anti-Gt/gust-type G protein (CytoSignal, #TF15) antibodies were commercially obtained. Antibodies against iguana parapinopsin and parietopsin were previously generated [10 (link)].

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Wada S., Kawano-Yamashita E., Sugihara T., Tamotsu S., Koyanagi M, & Terakita A. (2021). Insights into the evolutionary origin of the pineal color discrimination mechanism from the river lamprey. BMC Biology, 19, 188.

Publication 2021

pMAL Protein Fusion and Purification System

Amino acids Animals Anti antibody Antibodies Antigens Antiserums Chicken G protein Guinea pig Gust Iguana Immunohistochemistry Lamprey Mouse Protein Proteins region β arrestin

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Variable analysis

independent variables

The C-terminal 55-amino-acid region of the lamprey parietopsin protein
The C-terminal 50-amino-acid region of the lamprey parapinopsin protein
269 amino acids (V29–E297) of the β-arrestin protein

dependent variables

Immunohistochemistry results using the anti-parietopsin, anti-parapinopsin, and anti-β-arrestin antibodies

control variables

The pMAL Protein Fusion and Purification System (New England Biolabs) was used to prepare the antigens, following the previously reported methods [10]
Antibodies against iguana parapinopsin and parietopsin were previously generated [10]

positive controls

The anti-VGLUT (Synaptic Systems, #135 503), anti-Go-type G protein (MBL, #551), and anti-Gt/gust-type G protein (CytoSignal, #TF15) antibodies were commercially obtained

negative controls

Not explicitly mentioned

Annotations

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