Amersham ecl rainbow marker full range

Frozen eyes were lysed with ice-cold radioimmunoprecipitation (RIPA) buffer containing protease and phosphatase inhibitors. Sonicated lysate was centrifuged (12,000 g, 4 °C, 15 min), and the supernatant was collected. Eye homogenates were mixed with 2-mercaptoethanol and resolved by SDS-PAGE and transferred to activated PVDF membranes. The PVDF membranes were blocked with 2.5% skim milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature. Phospho-CREB primary antibody (1:1,000) (#9189; Cell Signaling Technology, Danvers, MA, USA), dissolved in 5% bovine serum albumin in TBST, was incubated 16 h at 4 °C. Membranes were washed with TBST. Secondary rabbit antibody (1:2000), dissolved in 2.5% skim milk in TBST, was incubated for 1 h at room temperature. Washed membranes with TBST was incubated with ECL prime (GE Healthcare, Chicago, IL, USA) for 15 min at room temperature and imaged on a LAS3000 (Fuji Film, Tokyo, Japan). ACTB primary antibody (1:1,000) (sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA) was used as an internal control after pCREB signals were eliminated by incubation in H₂O₂. The sizes of the protein were determined by Amersham ECL Rainbow Marker-Full Range (GE Healthcare). Quantification of immunoblots was conducted with Image J (https://imagej.nih.gov/ij/index.html).