Kwikquant ultra digital ecl

For cell lysis, we used 1% SDS lysis buffer (50 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl, 5 mM DTT, and 1% SDS) containing protease and phosphatase inhibitors (Thermo Fisher Scientific). Protein lysates were resolved in Novex Bolt 4–12% Bis-Tris gels (Life Technologies/Thermo Fisher Scientific) and transferred to nitrocellulose membranes. All membranes were blocked with 5% milk in TBS-T (Tris-buffered saline with 0.1% Tween) for 1 hour at room temperature and incubated overnight at 4C with various primary antibodies diluted in TBS-T plus 3% BSA (see supplemental materials), washed 3 times in TBS-T, then incubated with horseradish peroxidase-conjugated secondary antibodies (Kindle Biosciences #K1005, #K1006) and developed using KwikQuant Ultra Digital ECL (Kindle Biosciences #K1002). Luminescent signals were detected using a FujiFilm X-A2 digital camera. Raw images were then converted to black and white using Adobe Photoshop CS6 V13.0.6.

Cells were lysed in 1% SDS lysis buffer (50 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl, 5 mM DTT, and 1% SDS) containing protease and phosphatase inhibitors (ThermoFisher). Lysates (10–20 ug) were resolved in Bolt® 4–12% Bis-Tris Plus gels (ThermoFisher) and MOPS buffer, transferred to nitrocellulose membranes (GE Healthcare), then incubated overnight at 4°C with primary antibodies to BCL-6 and β-actin (see Supplementary Table S3). After washing and adding horseradish peroxidase-conjugated secondary antibodies (Kindle Biosciences) assays were developed using KwikQuant Ultra Digital ECL (Kindle Biosciences). Luminescent signal was detected using a FujiFilmX-A2digital camera and raw images converted to B/W with Adobe Photoshop CS6 V13.0.