Donkey anti mouse irdye 800 cw secondary antibody

Cultured and treated ARPE-19 cells were lysed in 1× Laemmli-SDS sample buffer plus protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Danvers, MA). Thirty micrograms of total protein, boiled at 95°C for 5 minutes, was used in each sample. The protein lysates were separated on a 4% to 12% gradient SDS-PAGE gel and transferred onto nitrocellulose membranes. Blocking was achieved by incubation for 1 hour in Odyssey blocking buffer (Licor Biosciences, Lincoln, NE) with 0.1% Tween-20. Membranes were incubated overnight at 4°C with 1:200 mouse anti–TYRP1 antibody (Santa Cruz Biotechnology). After washes, membranes were incubated with donkey anti-mouse IRDye^® 800CW secondary antibody (Licor) at 1:7500 dilution. Anti–GAPDH antibody (Cell Signaling) was used as loading control. Bands were detected with a Licor Odyssey imager. Densitometry analysis for relative abundance was performed with ImageJ software version 1.46r.

Relative expression of LuxS was determined by quantitative Western blot analysis of whole cell lysates. The total protein in the lysates was determined using the BCA Protein Assay kit (Thermo Scientific, Australia). Bands on Western blots were detected uisng anti-LuxS polyclonal murine antiserum at a dilution of 1:2000 and donkey anti-mouse IRDye 800 CW secondary antibody (LI-COR Biosciences, USA) at a dilution of 1:50000. The blot was scanned and LuxS expression was quantified using the Odyssey Infrared Imaging System (LI-COR Biosciences).