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ECL Western Blotting Substrate kit

Manufactured by AmyJet Scientific
Sourced in China

The ECL Western Blotting Substrate kit is a laboratory product designed for the detection and visualization of target proteins in Western blot analyses. The kit contains the necessary reagents to facilitate the chemiluminescent detection of protein bands on a membrane.

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2 protocols using ECL Western Blotting Substrate kit

Protein was extracted from HT29 cells using ice-cold RIPA buffer (Elabscience Biotechnology, Inc.) and determined by using a BCA protein assay kit (Phygene). Protein samples were subjected to separation via 10% SDS-PAGE (DetaiBio Tech) and transferred onto PVDF membranes (Roche Diagnostics), after which time the membranes were blocked with 5% non-fat milk for 2 h at room temperature. The membranes were incubated at 4˚C overnight with the following primary antibodies (all Abcam): Nrf2 (1:1,000, cat. no. ab137550), heme oxygenase-1 (HO-1; 1:2,000, cat. no. ab52947), NADPH dehydrogenase quinone 1 (NQO1; 1:10,000, cat. no. ab80588), GPX4 (1:1,000, cat. no. ab125066), ferritin heavy chain 1 (FTH1; 1:1,000, cat. no. ab183781), transferrin (1:1,000, cat. no. ab277635) and GAPDH (1:1,000, cat. no. ab8245). Following primary antibody incubation, the membranes were incubated with HRP-conjugated secondary antibodies (goat anti-mouse IgG H&L, 1:2,000, cat. no. ab6789; or goat anti-rabbit IgG H&L, 1:2,000, cat. no. ab6721) for 2 h at room temperature. An ECL Western Blotting Substrate kit (AmyJet Scientific, Inc.) was applied to visualize protein bands. The resultant images were analyzed using ImageJ software (version 1.8.0; National Institutes of Health) and the quantification of each group was performed in triplicate.
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Protein were separated by SDS-PAGE, and then transferred onto polyvinylidene difluoride membrane. The membrane was blocked, and probed with primary antibodies: anti-Bax and anti-Bcl-2 (1:2000; Abcam, Cambridge, MA, USA), anticleaved caspase-3 and anti-GAPDH (1:2500; Abcam), anti-p65 and anti-p-p65 (1:3000; Abcam), anti-IκBα and anti-p-IκBα (1:3500; Abcam), anti-Keap1 and anti-HO-1 (1:4000; Abcam), anti-Nuclear Nrf2, and anti-Lamin B1 (1:4500; Abcam). Followingly, the protein blots in the membrane were incubated with the corresponding secondary antibody (1:5000; Abcam) and visualized by ECL Western blotting Substrate Kit (AmyJet Scientific, Wuhan, China).
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