Active/Pro-Caspase 3 Monoclonal Antibody (31A1067)

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	-Mouse (1:500)

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-Mouse (1:500)

Publication protocol

Cells cultured in T-25 flasks were treated with 100 μM esculetin for different time intervals. They were lysed in RIPA buffer (50 mM Tris, 2 mM EDTA, 150 mM NaCl, 1 % Tergitol, 0.5 % sodium deoxycholate, 1 mM PMSF and 1X Protease Inhibitor) for 45 min at 4 °C and centrifugated at 13,000 g for 20 min at 4 °C. Total protein obtained as supernatant was determined using BCA protein estimation kit (Bangalore Genei). For collection of separate nuclear and cytoplasmic extract, NE-PER™ Nuclear and Cytoplasmic Extraction kit (Life Technologies, USA) was used as per manufacturer’s protocol. Equal amount of proteins (30–50 μg) were boiled in Laemmli buffer (2 % SDS, 10 % glycerol, 60 mM Tris-Cl pH 6.8, bromophenol blue 0.02 %) and separated on (8–12 %) SDS- polyacrylamide gels (SDS-PAGE) and transferred on polyvinyldifluoride (PVDF) membrane (Millipore, USA). Bovine Serum Albumin (BSA), 5 %, was used to block the PVDF membrane, followed by incubation with desired primary antibody for 3 h and horseradish peroxidase (HRP) conjugated secondary antibody for 45 min at 37 °C with intermittent washing thrice with 0.05 % tween-20 in PBS (v/v) at room temperature for 15 min each. Immunoreactive bands were probed with the enhanced chemiluminiscence (ECL) western blot detection system (Biogene, India) according to manufacturer’s instructions and viewed in gel documentation system LAS4000 (FUJIFILM, USA). Antibodies against caspase 3 (MA191637), caspase 8 (MA141280), caspase 9 (MA112562) and cytochrome C (MA5-11674) were purchased from Pierce (Thermo Scientific, USA). Antibody against phosphorylated Nrf2 (ab76026) was purchased from abcam. Antibody against proliferating cell nuclear antigen (PCNA) (ab18197) and NF-κB (sc-372) were provided as a kind gift by Prof Vani Brahmachari and Prof K Natrajan respectively from Dr. B.R Ambedkar Centre for Biomedical Research, University of Delhi. Antibody against inhibitor kappa B (IκB) (sc-847) was a kind gift by Prof Alok C Bharti from Department of Zoology, University of Delhi. Antibody against β actin (sc-47778), Nrf2 (sc-13032) and KEAP1 (sc-15246) along with all HRP conjugated secondary antibodies against rabbit, mouse and goat were purchased from Santa Cruz (USA).

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Manufacturer protocol

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