Quantification of Retinal Rhodopsin Levels

The levels of rhodopsin (RHO) were assessed in protein lysates from PW7 P23H and WT mouse retinas using a RHO antibody (1:1000, MABN15, Millipore, USA) in 5% nonfat milk (#M0841, Labscientific, USA) overnight at 4 °C^{8 (link),12 (link)}. The signals were detected using 1:2500 dilutions of corresponding horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (ECL) reagent (#32106, Thermo Scientific, USA), and then the digital images were visualized with an Azure Biosystems instrument. β-ACTIN (1:5000, A1978, Sigma, USA) was used as an internal control.

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Tomita Y., Qiu C., Bull E., Allen W., Kotoda Y., Talukdar S., Smith L.E, & Fu Z. (2021). Müller glial responses compensate for degenerating photoreceptors in retinitis pigmentosa. Experimental & Molecular Medicine, 53(11), 1748-1758.

Publication 2021

MABN15 β-ACTIN

Actin Antibodies Antibody Azure Chemiluminescence Dilutions Horseradish peroxidase Milk Mouse Protein Retinas Rhodopsin

Corresponding Organization : Boston University

Other organizations : Harvard University, Beth Israel Deaconess Medical Center, Merck & Co., Inc., Rahway, NJ, USA (United States)

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Variable analysis

independent variables

Mouse retina genotype (PW7 P23H and WT)

dependent variables

Levels of rhodopsin (RHO) protein

control variables

Anti-rhodopsin antibody (1:1000, MABN15, Millipore, USA)
5% nonfat milk (#M0841, Labscientific, USA) for blocking
Overnight incubation at 4 °C
Horseradish peroxidase-conjugated secondary antibodies (1:2500 dilution)
Enhanced chemiluminescence (ECL) reagent (#32106, Thermo Scientific, USA) for signal detection
β-ACTIN (1:5000, A1978, Sigma, USA) as an internal control

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