The cells were lysed on ice for 10 min with RIPA lysis buffer (P0013C, Beyotime, Shanghai, China) containing 1× Protease Phosphatase Inhibitor Cocktail (P1050, Beyotime) before being centrifuged at 13,000 g for 10 min at 4 °C. Total protein samples (40 µg) were resolved on 8% SDS-PAGE gel and transferred onto PVDF membranes. The membranes were blocked with 5% nonfat milk in PBST (0.05% Tween 100 in PBS), incubated with specified primary antibodies overnight at 4 °C, washed and incubated with a horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA), and then washed and detected with an enhanced chemiluminescence substrate (Roche, Indianapolis, IN, USA).
Enhanced chemiluminescence substrate
Manufactured by Roche
Sourced in United States
Enhanced chemiluminescence substrate is a laboratory reagent used to detect and quantify proteins in Western blot analysis. It generates a luminescent signal when exposed to the enzyme horseradish peroxidase, which is commonly used to label target proteins in this technique. The intensity of the luminescent signal is proportional to the amount of the target protein present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Protease phosphatase inhibitor cocktail, by Beyotime (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Sds polyacrylamide precast gels, by Bio-Rad (1 mentions)
Sc 8008, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
2 protocols using enhanced chemiluminescence substrate
1
Western Blot Protein Analysis Protocol
2
Protein Extraction and Western Blotting
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For total proteins extraction, cultured cells’ pellets were homogenized in a stringent SDS-containing RIPA buffer containing protease inhibitors as described previously [23 (link)]. Protein concentration was determined using the Lowry assay (DC protein assay kit; Bio-Rad, Hercules, CA, USA). The protein-containing lysate (15 μg) was run on 10% SDS-polyacrylamide precast gels (Bio-Rad) and electrotransferred onto nitrocellulose membranes (Bio-Rad). After an overnight blocking (1% reagent; Roche Diagnostics), the membranes were probed with mouse monoclonal antibodies directed to NFκB p65 (SC 8008, 1:275, Santa Cruz), or β-actin (1:10,000, Sigma Aldrich) or with the polyclonal antibody MxA (1:1000, Sigma Prestige Antibodies), followed by horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibody (1:2000, Santa Cruz and 1:10,000, Jackson, respectively). The immunoreactive proteins were detected on films using an enhanced chemiluminescence substrate according to the manufacturer’s instructions (Roche Diagnostics).
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