Protein lysates were prepared using RIPA buffer containing 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM NaF, and 30 µL/mL aprotinin. The proteins (20–30 µg) were resolved in SDS-PAGE, electro-transferred onto nitrocellulose membranes (Hybond-ECL, GE Healthcare) and blocked with 5% BSA. Primary antibodies included NRF2 (D1Z9C), BiP/GRP78 (C50B12), IRE1α (14C10), CHOP (L63F7), Calnexin (C5C9) and PERK (D11A8) from Cell Signaling Technologies; p-IRE1α (Ser724; NB100-2323) from Novus Biologicals; GCLC (Ab41463), beta-actin (Ab8227), TBP (ab818) and ATF6 (Ab37149) from Abcam; p-PERK (Thr 981, sc-32577), NQO1 (C-19), and ATF3 (C19) from Santa Cruz. After secondary antibody incubation (1:3000 in TBS-T, 2 h), the proteins were detected using Lumiglo substrate (Cell Signaling Technology, CA) and X-ray films. Immunoblot images are representative of three independent experiments.
Lumiglo substrate
Manufactured by Cell Signaling Technology
LumiGLO is a chemiluminescent substrate solution that is used to detect and quantify proteins in Western blotting. It generates a luminescent signal in the presence of specific enzymes, such as horseradish peroxidase (HRP), which can be detected and measured using a luminometer or film.
Automatically generated - may contain errors
Lab products found in correlation
Atf3 c19, by Santa Cruz Biotechnology (2 mentions)
Hybond ecl, by GE Healthcare (2 mentions)
P ire1α ser724 nb100 2323, by Novus Biologicals (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti phospho erk 197g2, by Cell Signaling Technology (1 mentions)
Beta actin ab8227, by Abcam (1 mentions)
Chop l63f7, by Cell Signaling Technology (1 mentions)
3 protocols using lumiglo substrate
1
Western Blot Analysis of UPR Proteins
2
Western Blot Analysis of Ras Signaling
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Cells were lysed with lysis buffer (50 mM Tris-HCl [pH7.5], 150 mM NaCl, 10 mM MgCl2, 0.5% Nonidet P-40) containing protease and phosphatase inhibitor cocktail (Thermo Scientific). Antibodies used for Western blotting are as follows: anti-phospho-ERK (197G2, Cell Signaling) anti-Ras (Ras-10, Millipore) anti-Rasal3 (homemade poly-clonal Rabbit IgG). Horseradish peroxidase-conjugated anti-IgG secondary antibodies against rabbit or mouse IgG (GE) were used with Lumiglo substrate (Cell signaling).
3
Protein Expression Analysis Protocol
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Protein lysates were prepared using RIPA buffer containing 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM NaF, and 30 μL/mL aprotinin. The proteins (20–30 μg) were resolved in SDS-PAGE, electro-transferred onto nitrocellulose membranes (Hybond-ECL, GE Healthcare) and blocked with 5% BSA. Primary antibodies (1:1000 dilution) included NRF2 (D1Z9C), BiP/GRP78 (C50B12) and CHOP (L63F7) from Cell Signaling; beta-actin (ab8227) from Abcam and ATF3 (C19) from Santa Cruz. After secondary antibody incubation (1:3000, 2 h), the proteins were detected using Lumiglo substrate (Cell Signaling Technology, CA).
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