Mini protean any kd acrylamide gel

Whole cell lysates were prepared in whole cell extract buffer (WCEB: 50 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, and complete protease inhibitor (Promega, Madison, WI)). Equal amounts of protein (30–50 μg) were electrophoresed on a mini-PROTEAN any KD acrylamide gel (Bio-Rad Laboratories, Hercules, CA) and transferred to Hybond ECL nitrocellulose (GE Healthcare, Chicago, IL). Transfer was verified via Ponceau S staining then blot was blocked with 5% nonfat dry milk (LabScientific, Highlands, NJ) in Tris buffered saline with 0.1% Tween 20 (TBST) for one hour at room temperature, followed by primary antibody in blocking buffer overnight at 4° C. After washing extensively with TBST, blots were incubated for 1–2 hours at room temperature with appropriate anti-mouse (GE Healthcare), anti-rabbit (GE Healthcare), or Rabbit anti-goat (Novus Biologicals, Littleton, CO), washed again with TBST, detected using Bio-Rad Clarity Western ECL Substrate (Bio-Rad Laboratories), and imaged via _MYECL Imager (Thermo Scientific). Primary Antibodies used and their concentrations can be found in Supplementary Table 3.

Whole-cell lysates were prepared in whole-cell extract buffer (WCEB: 50 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, and complete protease inhibitor [Promega]). Equal amounts of protein (30–50 μg) were electrophoresed on a mini-PROTEAN any KD acrylamide gel (Bio-Rad Laboratories) and transferred to Hybond ECL nitrocellulose (GE Healthcare). The transfer was verified via Ponceau S staining, then the blot was blocked with 5% nonfat dry milk (LabScientific) in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at room temperature, followed by primary antibody in blocking buffer overnight at 4°C. After washing extensively with TBST, blots were incubated for 1–2 h at room temperature with appropriate HRP-linked secondary antibody (GE Healthcare), washed again with TBST, developed using Pierce ECL Western Blotting Substrate (ThermoFisher Scientific), and exposed to film for detection. Primary Antibodies used and their concentrations can be found in Supplemental Table 3.

Whole-cell lysates were prepared in whole-cell extract buffer (WCEB: 50 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1%SDS, and complete protease inhibitor [Promega]). After sonication, equal amounts of protein (30–50 μg) were electrophoresed on a mini-PROTEAN any KD acrylamide gel (Bio-Rad Laboratories) and transferred to Hybond ECL nitrocellulose (GE Healthcare). The blot was blocked with 5% non-fat dry milk (LabScientific) in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 hour at room temperature, followed by primary antibody incubation in blocking buffer overnight at 4°C. After washing extensively with TBST, blots were incubated for 1–2 hour at room temperature with appropriate HRP-linked secondary antibody (GE Healthcare), washed again with TBST, developed using Bio-Rad Clarity Western ECL Substrate (Bio-Rad Laboratories), and imaged via MYECL Imager (Thermo Scientific). Antibody details including working dilutions can be found in S1 Table.