Enhanced chemiluminescence ecl method

Total kidney tissue protein was obtained with RIPA buffer as previously described. Protein concentrations were determined with a Bio-Rad protein assay kit (Hercules, CA, USA). For the western blot analysis, aliquots of the lysate containing 30–50 μg protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred onto a nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA). The membranes were subjected to immunoblot analysis and the proteins were visualized by an enhanced chemiluminescence (ECL) method (GE Healthcare, Buckinghamshire, UK). The cell lysates were separated by 12% SDS-PAGE, transferred onto a polyvinylidene fluoride membrane (GE Healthcare, Buckinghamshire, UK), blocked with 5% skimmed milk and hybridized with primary antibodies (diluted 1:1000). The antibodies against NF-κB-p65, IκB-α, TGF-β1, Fas and FasL were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The blots were then incubated with the horseradish peroxidase—conjugated secondary antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) for 1 h at room temperature. The blots were washed three times with PBS-T and then developed by enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL, USA) [12 (link)].

Cultured PC 12 cells were starved for 5 hours and then challenged with NGF, quercetin, and extract from Ginkgo Folium for 0, 5, or 10 min. Cells were solubilized in lysis buffer containing 125 mM Tris-hydrochloride (pH 6.8), 4% sodium dodecyl sulfate (SDS), 20% glycerol, and 2% 2-mercaptoethanol and stored frozen at −20°C. Lysates were separated on 8% SDS-polyacrylamide gels and transferred to a nitrocellulose membrane. The membrane was blocked with 5% nonfat milk and then incubated overnight with anti-phospho-Erk-1/2 primary antibodies (1 : 1,000; Cellular Signaling Technology, Danvers, MA), followed by anti-rabbit secondary antibodies (1 : 5,000; Invitrogen Life Technologies) for an hour. The immune complexes were visualized by the enhanced chemiluminescence (ECL) method (GE Healthcare, Chicago, IL). The samples were run on the same gel under strict standardized ECL conditions and the intensities of the bands were compared using an image analyzer and ImageJ 1.48 v software [16 (link)].

Total colon tissue protein was obtained with RIPA buffer as described [33 (link)]. Protein concentrations were determined with a Bio-Rad protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). For the western blot analysis, aliquots of the lysate containing 30–50 μg protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred onto a nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA). The membranes were subjected to immunoblot analysis and the proteins were visualized by an enhanced chemiluminescence (ECL) method (GE Healthcare). The cell lysates were separated by 12% SDS-PAGE, transferred onto a polyvinylidene fluoride membrane (GE Healthcare), blocked with 5% skimmed milk and hybridized with primary antibodies (diluted 1:1000). The antibodies against Bax, Bcl-2, caspase-3, caspase-9, NF-κB, IκB-α, iNOS and COX-2 were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), then incubated with the horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) for 1 h at room temperature. The blots were washed three times with PBS-T and then developed by enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL, USA).