caspase-8 Antibody (1.1.40): sc-81656

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:200) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:200) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

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.).

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Manufacturer protocol

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