Tris hcl sds polyacrylamide gels

Tissues were collected and lysed in RIPA buffer (Sigma, St. Louis, MO, USA) in the presence of protease and phosphatase inhibitors (Halt™ protease and phosphatase inhibitor, Thermo Scientific, Waltham, MA, USA), and samples were prepared in protein sample buffer (0.25% bromophenol blue, 0.5M dithiothreitol, 50% glycerol, 10% sodium dodecyl sulfate (SDS), 0.25M Tris-Cl pH 6.8, and trace amounts of bromophenol blue), boiled, and stored at −80 °C. For gel electrophoresis and Western blot analyses, samples were run on 4–15% precast Tris-HCl SDS-polyacrylamide gels (BioRad, Hercules, CA, USA) and transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, Burlington, MA, USA). Blots were successively probed with anti-COX4 (Cell signaling technology, #11967S), -CRIF1 (Santa Cruz, #sc-374122), -caspase 8 (Cell signaling technology, #4790S), -caspase 9 (Cell signaling technology, #9508S), -BCL-2 (Cell signaling technology, #2876S), or -β-actin (Cell signaling technology, #4967S) antibodies at 1:1000 dilutions in TBS containing 3% protease free BSA (Sigma, St. Louis, MO, USA). Blots were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Burlington, MA, USA) and the images were acquired and quantitated using Azure 300 Chemiluminescent Western Blot Imaging System (Azure Biosystems, Dublin, CA, USA).

Tissues were collected and lysed in RIPA buffer (Sigma) in the presence of protease and phosphatase inhibitors (Thermo Scientific), and samples were prepared in protein sample buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate, 1% β-mercaptoethanol and trace amounts of bromophenol blue), boiled, and stored at -80°C. For gel electrophoresis and western blot analyses, samples were run on 4–15% precast Tris-HCl SDS-polyacrylamide gels (BioRad) and transferred to polyvinylidenedifluoride (PVDF) membranes (BioRad). Blots were successively probed with anti-COX4 or -actin antibodies at 1∶1000 dilutions in TBS containing 3% protease free BSA (Sigma). Blots were visualized using SuperSignal chemiluminescence kits (Pierce Biotechnology) and the images were acquired and quantitated using FluorChem Q (Alpha Innotech).

Protein samples were separated on pre-cast 10% Tris.HCl SDS-polyacrylamide gels (BioRad) and transferred to Immobilon-P membranes (Millipore) using a semi-dry blotter. For 10 well gels each sample loaded represented 5×10⁴ cells. Following transfer, filters were blocked for one hour at room temperature using PBS containing 0.1% v/v Tween-20 (PBS-T), 5% w/v milk powder. Primary antibodies were used at a dilution of 1∶5000 in PBS-T, 5% w/v milk powder. Following several washes with PBS-T the membranes were incubated for 1 hour with the appropriate HRP-conjugates and the bound antibodies detected by BM chemiluminescence (Roche). Bands were imaged using a Syngene G:Box chemiluminescence imager and the image file processed for pixel density by Genetools software (Syngene).