Enhanced chemiluminescence ecl detection kit

Leaf disks from two fresh and fully expanded leaves of three independently grown Nt-PTOX-OE plants were ground into fine power in liquid nitrogen. Total soluble proteins (TSP) and membrane proteins (MP) were extracted and the protein concentration was determined as described earlier (Ahmad et al., 2012 (link)). Proteins were loaded on an equal protein concentration basis for TSP and equal chlorophyll content basis for the MPs, separated by 12.5% (w/v) SDS-PAGE, transferred to a nitrocellulose membrane using an iBlot^® Dry Blotting System (Invitrogen, United States) and probed using either anti-HA (Human influenza hemagglutinin) tag antibodies or D1 antibodies (Roche Applied Science, Germany). Broad-range pre-stained multicolor molecular weight standards, Spectra^TM (Fermentas, United States), were run alongside samples to determine the sizes of protein bands. The gels were stained with Coomassie Brilliant Blue ‘R-250.’ Proteins were immuno-detected using horseradish peroxidase (HRP) conjugated to secondary antibodies raised against rabbit IgG and an enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia, United Kingdom) with the signal captured by X-ray film (Kodak, United States).

Dulbecco’s modified essential medium, fetal bovine serum (FBS), penicillin and streptomycin were supplied by Gibco (Carlsbad, CA). The enhanced chemiluminescence (ECL) detection kit was purchased from Amersham Biosciences (Piscataway, NJ). 3-[4, 5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) and other chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless specified. The following antibodies were used: anti-GRP78, anti-GRP94, anti-calpain I and anti-calpain II (Santa Cruz Biotechnology, Santa Cruz, CA); anti-caspase 3, (Upstate Biotechnology, Charlottesville, VA); anti-PARP (BD Pharmingen, San Jose, CA); anti-β-actin (Sigma-Aldrich, St. Louis, MO).

NGEN (2,3‐dihydro‐5,7‐dihydroxy‐2‐[4‐hydroxyphenyl]‐4H‐1‐benzopyran‐4‐one), dithiothreitol (DTT), 4′,6‐diamindino‐2‐phenylinodole (DAPI), EGTA, leupeptin, and Triton X‐100 were bought from Sigma‐Aldrich Co. (St Louis, MO). Anti‐Bcl‐2 (sc‐7328), anti‐Bcl‐xL (sc‐7195), anti‐Bad (sc‐7869), and anti‐Bax (sc‐493) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti‐cytochrome c (Cat. No. 556432) was bought from PharMingen (San Diego, CA). Anti‐β‐Actin (A5441) was purchased from Sigma‐Aldrich Co. and anti‐PUMA (Cat. No. PC686) was bought from Oncogene Science, Inc. (Uniondale, NY). Anti‐cytochrome oxidase IV (Cat. No. 556423) was purchased from Molecular Probes (Eugene, OR). Secondary antibodies of peroxidase‐conjugated anti‐rabbit IgG (Cat. No. NA934), anti‐mouse (Cat. No. NA931) and the enhanced chemiluminescence (ECL) detection kits were obtained from Amersham Life Science (Buckinghamshire, UK). Inhibitors of Caspase‐3 (z‐DEVD‐fmk), caspase‐8 (z‐IETD‐fmk), and caspase‐9 (z‐LEHD‐fmk) were obtained from KAMIYA Biomedical Company (Seattle, WA). Reagents of caspase activity assay were purchased from R&D Systems (Minneapolis, MN). All other chemicals with analytical grade quality had been obtained through commercial sources.

HeLa and Vero cell lines, including a control group, were subjected to trypsinization for cell collection, subsequent to treatment with siRBBP6 and cisplatin (CDDP). Total protein extracts from both treated and untreated cells were obtained using a RIPA buffer (Thermofisher, 3747 N. Merdian Rd. RockFord, IL61101. Waltham, MA, USA). Following this, 20 µg of the protein extracts were separated using SDS-PAGE (Bio-Rad laboratoty Inc., Pretoria, South Africa), and were transferred onto polyvinylidene difluoride membranes using the transfer tank system, also from Bio-Rad. Subsequently, membranes were incubated with primary antibodies and left overnight in a −4 °C refrigerator; this was followed by a 1 h incubation with secondary antibodies the next day, and the specific antibodies used are listed in Table 3. Enhanced chemiluminescence (ECL) detection kits from Amersham Life Science, California city, CA, USA, were used to identify the proteins of interest, and image capture and analysis were performed using the ChemidocTM MP imaging system from Bio-Rad.

Cells (5 × 10⁶) for sample preparation were harvested by centrifugation and washed in PBS (137 mM NaCl, 4 mM Na₂HPO₄, 1.7 mM KH₂PO₄, 2.7 mM KCl). Pellets were resuspended in urea cracking buffer (6 M urea, 10 mM Na₂HPO₄, 1% β-mercaptoethanol, pH 7) and boiled after the addition of loading sample buffer (67.5 mM Tris-HCl, pH 6.8, 3% SDS, 10% glycerol, 5% β-mercaptoethanol). SDS-PAGE was performed, and polyvinylidene difluoride (PVDF) membranes were incubated with rabbit polyclonal anti-TbCDA (1:500) antibody generated against recombinant T. brucei CDA, rabbit polyclonal anti-TbdUTPase (1:75,000) antibody generated against recombinant T. brucei dUTPase, or mouse monoclonal anti-DCTD (1:1,000 [Santa Cruz Biotechnology]) or anti-β-tubulin (1:5,000 [Sigma]) antibodies. Bound antibodies were revealed by using goat anti-rabbit IgG (1:5,000) or goat anti-mouse IgG (1:3,000) antibodies (Promega) and the ECL enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech).