Tris glycine sds electrophoresis buffer

The protein profile of PPE, Ara h 1, Ara h 2, Ara h 3 and Ara h 6 was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing and reducing conditions. Each protein sample was dissolved in PBS (137 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1 mM KH₂PO₄, pH 7.2) and mixed 1:1 (v/v) with 2x Laemmli sample buffer (161-0737, Bio-Rad, Hercules, CA, US) with the addition of 2 M dithiothreitol (348-12-3, Sigma-Aldrich, Darmstadt, Germany) for reducing conditions, and subsequently heated for 5 min at 95°C. Five micrograms of protein/well as well as 3 µL of molecular marker (161-0363, Bio-Rad) were loaded onto Mini Protean TGX Precast Protein 4-20% Gels (4568094, Bio-Rad) and electrophoresed on a Mini-PROTEAN Tetra Cell (Bio-Rad) filled with 10X Tris/Glycine/SDS electrophoresis buffer (161-0732, Bio-Rad) (1:10 v/v) prepared according to manufacturer’s protocol. The gels were stained with Coomassie Blue (161-0786, Bio-Rad) at room temperature (RT) for 3 h, and subsequently destained with MQ water at RT overnight. Destained gels were photographed using Imager ChemiDoc XRS+ (Bio-Rad).

The crude protein extracts (20 μg) were denatured in sample loading buffer and separated using electrophoresis in a polyacrylamide gel system (4–20% Mini-PROTEAN^® TGXTM Precast Gels with Mini-PROTEAN^® Tetra Cell using Tris/Glycine/SDS Electrophoresis Buffer (Bio-Rad, Hercules, CA, USA). The separated proteins were transferred onto Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA) in Tris/glycine buffer with Mini Trans-Blot^® Cell (Bio-Rad, Hercules, CA, USA).

Cultured SMCs were washed with ice cold PBS twice, and 80 μl Laemmli sample buffer (60 mM Tris-Hcl, pH 6.8, 10% glycerol, 2% SDS) was added. After lysis and protein determination using a detergent-compatible protein assay from Bio-Rad (500–0116), bromophenol blue (0.01%) and β-mercaptoethanol (5%) were added to the remainder of the lysates. ≈20 μg protein was loaded per lane on Bio-Rad TGX Criterion gels and proteins were separated using Bio-Rad Tris/Glycine/SDS electrophoresis buffer at 200 V. Following separation, proteins were transferred to nitrocellulose (0.2 μm) using the Trans-Blot Turbo Transfer System (Bio-Rad) and western blotting was done essentially as described^{57 (link)}. Membranes were cut horizontally to allow for detection of multiple targets as needed, and hence blots covering the entire range of molecular weights are not available throughout. Full blots for all display items are available in the Supplementary Information file. The following primary antibodies were used: NEXN (Abcam, ab213628), P-YAP (Cell Signaling Technology,#4911), T-YAP (Cell Signaling Technology, #4912), HSP90 (BD Transduction Laboratories, 610418), calponin/CNN1 (Abcam, ab46794), SM22/TAGLN (Abcam, ab14106). Secondary anti-mouse and anti-rabbit antibodies were from Cell Signaling Technology (#7076 S, #7074 S).