Ecl horseradish peroxidase linked anti rabbit na934

To prepare whole cell lysates, ∼3 × 10⁵ cells were pelleted, washed once with 10 mM Tris, pH 7.4, and precipitated with 10% trichloroacetic acid (TCA). TCA precipitates were incubated on ice for 30 min, centrifuged (18,000 × g, 10 min, 4°C), washed with ice-cold acetone, centrifuged at 18,000 × g for 5 min at 4°C, and resuspended in 2× SDS–PAGE sample buffer. For Western blots, samples were resolved by SDS–PAGE and transferred to 0.45-μm polyvinylidene fluoride (PVDF) membranes (Thermo Scientific). Blots were blocked and probed as previously described (Turkewitz et al., 1991 (link)). The polyclonal anti-Grl1p serum and purified anti-GFP mAbs were diluted 1:2,000 and 1:5000, respectively. Protein was visualized with either ECL horseradish peroxidase–linked anti-rabbit (NA934) or anti-mouse (NA931; Amersham Biosciences, Little Chalfont, United Kingdom), secondary antibody diluted 1:20,000, and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).