Publication protocol
The cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer (KeyGen Biotech Co., Ltd.) for 30 min. Following centrifugation at 8,000 × g for 5 min at 4°C, the soluble proteins were boiled at 95–100°C for 5 min, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (KeyGen Biotech Co., Ltd.) at a 70 V constant voltage, and transferred electrophoretically onto polyvinylidene difluoride membranes (EMD Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk at room temperature for 1.5 h and immunoblotted at 37°C for 1.5 h with the following antibodies: Polyclonal goat anti-human CLN3 (cat. no. sc-49627; 1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), poly-clonal mouse anti-human cleaved caspase-8 (cat. no. sc-81656; 1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), polyclonal rabbit anti-human Bcl-2 (cat. no. KG22169; 1:500; KeyGen Biotech Co., Ltd.), polyclonal rabbit anti-human Bax (cat. no. KG22165; 1:500; KeyGen Biotech Co., Ltd.), polyclonal rabbit anti-human cleaved PARP (cat. no. YC0073; 1:1,000; ImmunoWay Biotechnology, Newark, DE, USA) and poly-clonal rabbit anti-human cleaved caspase-3 (cat. no. KG22205; 1:200; KeyGen Biotech Co., Ltd.), followed by incubation with horseradish peroxidase-linked secondary antibody (1:10,000; KeyGen Biotech Co., Ltd.) at 37°C for 40 min. Finally, the protein bands were visualized using an enhanced chemiluminescence detection system (KeyGen Biotech Co., Ltd.).
Full paper
Login or
join for free to view the full paper.
Videos
Check out videos that might be relevant for performing Western blotting Caspase-8 using caspase-8 Antibody (1.1.40): sc-81656 from Santa Cruz Biotechnology. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.
We haven't found any additional videos for this experiment / product combination yet.