Phospho-eIF2α (Ser51) Antibody

Protocol tips

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	Dilute primary Ab at 1:1000 and incubate with primary antibodies overnight at 4°C

Protocol tips
Dilute primary Ab at 1:1000 and incubate with primary antibodies overnight at 4°C

Publication protocol

Cells were seeded at 400,000 cells per well in a six-well plate. Following the indicated treatments, cells were washed with PBS, and then attached cells were collected and lysed with radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% (w/v) SDS, 1% (v/v) NP40, and 0.5% (w/v) sodium deoxycholate] supplemented with protease inhibitors (Complete, Mini Protease Inhibitor Cocktail; Roche Diagnostics, Indianapolis, IN), phenylmethylsulfonyl fluoride (2 mM), and phosphatase inhibitors (Sigma-Aldrich). Cells were sonicated on ice and then centrifuged at 10,000g for 14 minutes at 4°C. The supernatants were collected, and protein concentrations were measured using the method of Lowry (Lowry et al., 1951). Proteins were diluted in 2× Laemmli SDS sample buffer and heated to 70°C for 5 minutes, and then separated on a 12% SDS-PAGE precast minigel (Bio-Rad Laboratories, Hercules, CA). A 7.5% SDS-PAGE precast minigel was used to separate the high molecular weight oligomers. Proteins were transferred to polyvinylidene fluoride membranes in 25 mM Tris and 192 mM glycine containing 20% (v/v) methanol at 4°C. Membranes were incubated in blocking buffer [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.2% (v/v) Tween 20 (TBST) containing 5% (w/v) nonfat dry milk] for 1 hour, and then incubated with primary antibodies overnight at 4°C. Membranes were washed every 10 minutes in TBST for 1 h and then probed with horseradish peroxidase–conjugated goat anti-rabbit or horseradish peroxidase–conjugated goat anti-mouse IgG (1:5000; Jackson Immunoresearch Laboratories, West Grove, PA) for 30 minutes at room temperature. Membranes were washed three times in TBST and protein bands were visualized using enhanced chemiluminescence (Thermo Scientific, Rockford, IL). Photoshop CS6 (Adobe, San Jose, CA) was used to quantitate immunoblots from three independent experiments

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for Phospho-eIF2α (Ser51) Antibody below.

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