Mice (male; 2 to 3 months of age; n = 6 per group) were acutely sacrificed by decapitation. The brain (the ACC) was then dissected quickly within 30 s, snap-frozen in liquid nitrogen, and subsequently lysed in radioimmunoprecipitation assay buffer containing 1% Triton X-100, 0.1% SDS, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktail (Sigma-Aldrich). The extracted proteins were subjected to SDS–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (0.45-μm pore, Millipore), which was incubated in a membrane-blocking agent (GE Healthcare) for 30 min and probed with anti-S6 ribosomal protein (rabbit, 1:1000; Cell Signaling Technology) or anti–phospho-S6 (Ser235/236) antibodies (rabbit, 1:1000; Cell Signaling Technology) and subsequently with horseradish peroxidase–conjugated anti-rabbit immunoglobulin G (1:10,000; GE Healthcare). Signals were detected with an ECL agent (GE Healthcare). Blotting images were acquired by ChemiDoc XRS imaging system (Bio-Rad), and the densitometric analysis was performed using ImageJ (NIH).
Ecl agent
Manufactured by GE Healthcare
ECL agent is a chemiluminescent detection reagent used in Western blotting analysis to visualize and quantify target proteins. It is a sensitive and reliable solution for protein detection and quantification in biological samples.
Automatically generated - may contain errors
2 protocols using ecl agent
1
Acute Sacrificing and Western Blot Analysis of S6 Ribosomal Protein in Mouse ACC
2
Western Blot Analysis of Extracellular Matrix Proteins
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For western blot analysis, conditioned media samples or whole cell lysate samples were subjected to SDS-PAGE and subsequently transferred to PVDF membranes (Millipore). Membranes were blocked in 5% milk for two hours prior to primary antibody Rabbit anti-TNC (Santa Cruz, 1:1000) incubation overnight. Tubulin was used as a control for protein loading and was detected using a mouse anti-α-tubulin antibody (1:5000, Sigma). Membranes were incubated with the peroxidase-coupled secondary antibodies for 1 hour. After extensive washing, the membrane was visualized using ECL agent (GE Healthcare Biosciences).
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