Goat anti rabbit igg h l hrp conjugate 1721019

Cells were seeded in a 24 plate (2x10⁵ per well) and incubated with or without amino acid at indicated concentration for the indicated time. Cells were washed with PBS and lysed by adding ice-cold 100 μL RIPA (Sigma) supplemented with Protease Inhibitor Cocktail (Roche). After 10 min incubation on ice, lysates were cleared by centrifugation (10 min x 24k r.p.m., 4 °C) and 20 μL 6× SDS sample buffer was added, and samples were incubated at 95 °C for 10 min. 10 μL were loaded per lane on a NuPAGE gel. Proteins were transferred using an iBlot onto a Nitrocellulose membrane, blocked with 5% milk powder/TBST and incubated with primary followed by secondary antibodies (Bio-Rad Goat Anti-Rabbit IgG (H+L)-HRP Conjugate #1721019; Goat Anti-Mouse IgG (H + L)-HRP Conjugate #1706516). Primary antibody was typically incubated over night at 4°C in 5% Milk/TBST, whereas secondary was incubated in TBST for 1h at RT. HRP signal was visualised using Illuminata Forte (Millipore) substrate and a ChemiDoc digital imager (Bio-Rad). For acetylation-specific antibodies, milk was replaced with purified BSA (SIGMA A9418). Acetylation-specific antibodies were used at 1:500 to 1:5,000 dilutions with consistent results.