Rats were killed and then their stomachs were cut along the greater curvature. Briefly, specimens from the pyloric area were extracted in lysis buffer, homogenized, and centrifuged at 12000 × g at 26750 rpm for 5 min. Mitochondria were isolated when needed. Total proteins were examined by a BCA kit (Beyotime, China). Subsequently, the equal proteins were separated by SDS/PAGE (10% gels) and transferred to PVDF membrane (Millipore, U.S.A.). After blocking with 5% fat-free milk, primary antibodies against caspases (-3, -8, and -9), cytochrome c, Fas, Bax, Bid, NF-κB p65, Ki67, or p53 (all purchased from Santa Cruz, California, U.S.A.) were added, followed by incubations with secondary antibodies horseradish peroxidase-conjugated IgG. GAPDH served as the loading control. As for NF-κB nuclear p65, Lamin B served as the loading control. Bands were visualized with chemiluminescence kit (Applygen Technologies, China) and quantitated using NIH ImageJ software.
Chemiluminescence kit
Manufactured by Applygen
Sourced in China
The Chemiluminescence kit is a laboratory equipment designed to detect and measure light-emitting chemical reactions. The kit provides the necessary components and reagents to perform chemiluminescence-based assays, which involve the emission of light as a result of a chemical reaction.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using chemiluminescence kit
1
Molecular Mechanisms of Rat Stomach Apoptosis
2
Western Blot Analysis of Recombinant C-NuMA
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The procedure of Western blot was described previously [20 (link)]. The purified recombinant proteins of C-NuMA were separated by SDS-PAGE and transferred to the polyvinylidene difluoride (PVDF) membrane. Blocked the membranes with 5% non-fat milk in PBS at 37°C temperature for 1 hours, the membranes were then incubated with 10 sera from BD patients (same group to immunofluorescence detection) and 10 healthy controls (1:1000 dilution) at 4°C temperature, overnight. After three times washing with 1% PBST buffer, the unbounded antibodies were removed. Then membranes were immersed with conjugated HRP-goat anti-human IgG (1:6000 dilutions, ImmunoHunt, Beijing, China) for 1 hour at 37°C temperature. Then, the membranes were detected by chemiluminescence kit (Applygen, Beijing, China).
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