Western Blot Protein Detection Protocol

Western blot was performed as previously described^{58 (link)}. Briefly, denatured proteins were resolved in 12% SDS-PAGE gels, transferred to nitrocellulose membranes, and immunoblotted with antibodies against each primary antibody as bellows; anti-flag antibody (1:1000 dilution, rabbit F7425 Sigma-Aldrich), anti-HA antibody (1:1000 dilution 16B12 mouse, 901514 Biolegend), anti-myc antibody (1:1000 dilution; 9E10 mouse, sc-40; Santa Cruz Biotechnology), anti-ß-arrestin1/2 antibody (1:1000 dilution; D24H9 rabbit; Cell signaling), Immunoreactivity was revealed using secondary antibodies coupled to 680 or 800 nm fluorophores (LI-COR Biosciences, Lincoln, NE, USA), and readings were performed with the Odyssey LI-COR infrared fluorescent scanner (LICOR Biosciences).

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Oishi A., Karamitri A., Gerbier R., Lahuna O., Ahmad R, & Jockers R. (2017). Orphan GPR61, GPR62 and GPR135 receptors and the melatonin MT2 receptor reciprocally modulate their signaling functions. Scientific Reports, 7, 8990.

Publication 2017

anti-flag antibody anti-HA antibody anti-myc antibody anti-ß-arrestin1/2 antibody D24H9 rabbit Odyssey LI-COR infrared fluorescent scanner

Anti antibody Antibodies Antibody Dilution Gels Membranes Mouse Nitrocellulose Proteins Rabbit Sds page Western blot

Corresponding Organization :

Other organizations : Institut Cochin, Inserm, Centre National de la Recherche Scientifique

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Variable analysis

independent variables

Antibodies used for immunoblotting: anti-flag antibody, anti-HA antibody, anti-myc antibody, anti-ß-arrestin1/2 antibody

dependent variables

Immunoreactivity levels detected using secondary antibodies coupled to 680 or 800 nm fluorophores and measured with the Odyssey LI-COR infrared fluorescent scanner

control variables

Denatured proteins resolved in 12% SDS-PAGE gels
Proteins transferred to nitrocellulose membranes

controls

No positive or negative controls were explicitly mentioned in the provided protocol.

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