Relative protein expression of Grk homologs in zebrafish cones using a novel zebrafish-specific anti-Grk7 antibody. (A) Immunodetection of 25 μg each of recombinant FLAG-tagged Grk7a and Grk7b prepared from HEK293 cells as described in “Methods.” For immunodetection, the immunoblot was incubated with anti-Grk7 (red) and anti-FLAG (green) antibodies at dilutions of 1:10,000 followed by incubation with secondary antibodies at a dilution of 1:15,000. (B) Immunodetection of endogenous Grk proteins in isolated retinal homogenates of adult zebrafish and heads of 5 dpf larvae. 40 μg of protein was loaded in each lane and the immunoblot was probed using a 1:10,000 dilution of the anti-Grk7 (red) and anti-Grk1 (green) antibodies followed by incubation with a 1:15,000 dilution of the secondary antibodies. (C) Ratio of Grk7 to Grk1 protein expression during zebrafish development. Samples were prepared, immunoblotting performed and quantification performed using Flag-tagged Grk1 and Grk7 as described in “Methods.”
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