Clarity max detection kit

Bacteria grown overnight in TGP containing 0.2 mM KH₂PO₄ (limited Pi concentration) were centrifuged, and a culture volume corresponding to an OD₆₀₀ of 1.0 (approx. 10⁹ cells) was resuspended in 0.1 ml Application Buffer (0.5 M Tris/HCl, 2% SDS, 5% 2-mercaptoethanol, 10%, v/v, glycerol and 0.01% bromophenol blue) and boiled for 5 min. Ten μl samples were resolved by standard SDS-PAGE (12.5% acrylamide). Following electrophoresis, proteins were transferred to a nitrocellulose membrane using a Trans-blot Semi-Dry Transfer Cell (BioRad, USA), as recommended by the manufacturer. The membrane was subjected to blocking with 5% skimmed milk and exposed to anti-FLAG M2 (Sigma) monoclonal antibodies, anti-RpoS (Neoclone) monoclonal antibodies (1,000X dilution) or anti-RpoD (Santa Cruz) monoclonal antibodies (5,000X dilution), followed by exposure to anti-mouse IgG serum conjugated to peroxidase (Thermo Scientific) diluted 10,000-20,000. Membranes were developed using the Clarity Max detection kit (Bio-Rad) and read in the Bio-Rad Imaging System.

For analysis of chemoresistance-related proteins, cells were exposed to 50 µM GQDs, MSN or PLA for 72 h and harvested by trypsinization (0.125% trypsin; Invitrogen). After three washes with PBS, a total of 2 × 10⁶ cells were lysed in Cell Extraction Buffer (Invitrogen, Carlsbad, CA, USA) and quantified using the Bio-Rad protein assay solution (Life Science Research, Hercules, CA, USA). A total of 20 µg proteins were subjected to 10% polyacrylamide gels and Western blotting was performed, as previously described [79 (link)]. Antibodies for XIAP (1:1000 dilution; Cell Signaling, MA, USA), Survivin (1:1000 dilution; Cell Signaling, MA, USA), cIAP-1 (1:1000 dilution; Cell Signaling,MA, USA), Bcl-2 (1:1000 dilution; DakoCytomation Denmark A/S, Produktionsvej Glostrup) and Bak (1:1000 dilution; Cell Signaling, MA, USA) were used. As a loading control, membranes were probed with the β-actin antibody (1:1000 dilution; Sigma-Aldrich, MO, USA). Mouse and rabbit secondary antibodies were purchased from. Primary antibodies were detected using horseradish peroxidase linked anti-rabbit (1:40,000 dilution; GE Healthcare^®, WI, USA) and anti-mouse (1:40000 dilution; GE Healthcare^®, WI, USA) conjugates and visualized using the Clarity Max™ detection kit (BioRad Laboratories, Hercules, CA, USA). Bands were detected using a Li-Cor imager (Biociences, Lincoln, NE, USA).