Anti-Caspase-8 antibody (ab25901)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.	-Rabbit (1:1000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Upstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.
Protocol tips
-Rabbit (1:1000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Publication protocol

The cells were plated into 6-well plates at a density of 2×105 cells per well. The following day, the cells were treated with CM (0.5, 1.0 and 2.0 mg/ml) for 24 h. The cells or tumor tissues were lysed using radioimmunoprecipitation assay buffer (Sigma-Aldrich) containing 1% protease inhibitor cocktail (Sigma-Aldrich) and 2% phenylmethanesulfonyl fluoride (Sigma-Aldrich). Tumor tissues were homogenized using a ZW-A trace vibrator (Ronghua Instrument Manufacturing Co., Ltd.). Lysates were centrifuged at 10,000 × g for 5 mins at 4°C and the concentration of protein was determined by the Coomassie brilliant blue method (Nanjing Jiancheng Biotechnology Co., Ltd., Nanjing, China) The proteins (30 µg) were separated on a 12% SDS-PAGE gel [materials obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China)] and transferred electrophoretically onto nitrocellulose membranes (0.45 µm; Bio Basic, Inc., Markham, ON, Canada). The transferred membranes were then blocked with 5% bovine serum albumin (Sigma-Aldrich) for 4 h at 4°C prior to blotting with the following primary antibodies at 4°C overnight, at a dilution of 1:1,000: Monoclonal rabbit anti-cleaved poly (ADP-ribose) polymerase (PARP; cat. no. ab32064; Abcam, Cambridge, UK), polyclonal rabbit anti-cleaved caspase-3 (cat. no. ab13847; Abcam), poly-clonal rabbit anti-cleaved caspase-8 (cat. no. ab25901; Abcam), monoclonal rabbit anti-Bax (cat. no. ab32503; Abcam) and polyclonal rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (cat. no. ABS16; EMD Millipore, Billerica, MA,USA). The membranes were subsequently incubated with horseradish peroxidase-conjugated mouse anti-rabbit secondary antibody (cat. no. sc-2357; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Chemiluminescence was analyzed using Amersham ECL Western Blotting Detection reagent (cat. no. RPN2106; GE Healthcare, Buckinghamshire, UK). The intensities of the bands were quantified by scanning densitometry using Image J software.

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Manufacturer protocol

Download the product protocol from Abcam for Anti-Caspase-8 antibody (ab25901) below.

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