Bolt mes sds running buffer

Leaves were harvested 2–3 days after infiltration with Agrobacterium suspensions, frozen in liquid nitrogen and stored until further processing at -80°C. All subsequent steps were performed at 4°C. Leaves were ground using mortar and pestle and proteins were extracted with lysis buffer (10 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.5 mM EDTA, 0.25% (v/v) Triton-X 100, 0.25% (v/v) Tween-20, 5 mM DTT, 4% (w/v) polyvinylpyrrolidone) supplemented with 2x protease inhibitor cocktail without EDTA (#11873580001, Roche) for 1 h at 4°C in an Eppendorf ThermoMixer at 2000 rpm. Lysates were centrifuged for 15 min at 10 000 g. The supernatant was separated on Bolt pre-mixed cast gradient gels 4–12% with Bolt Mes-SDS running buffer (ThermoFisher Scientific) according to the manufacturer’s instructions. Proteins were transferred for 100 min at 100 V to nitrocellulose membrane using Towbin buffer (25 mM Tris, 192 mM glycine, 20% (v/v) ethanol), detected with 1:2500 diluted anti-GFP HRP conjugated antibodies (Miltenyi Biotec Cat# 130-091-833, RRID:AB_247003, Lot no: 5180709500) using SuperSignal West Pico chemiluminescent substrate (ThermoFisher Scientific) and documented with a CCD-camera (ChemiDoc, Bio-Rad). Original Western blots are provided in Supplementary information S1 Raw images.

Samples were prepared as previously described [28 (link)], resolved on Bolt pre-cast 4-12% gels (Thermo Fisher Scientific), using Bolt MES SDS running buffer (Thermo Fisher Scientific) and transferred onto nitrocellulose membranes. After a blocking step in 5% non fat-dried milk in phosphate-buffered saline (PBS)-0.1% Tween, membranes were incubated with primary antibodies overnight at 4 °C. After three washes in PBS - 0.1% Tween, membranes were incubated with the appropriate HRP-linked secondary antibodies (Bio-Rad Laboratories) for 45 min at room temperature, washed with PBS-0.1% Tween and analysed by chemi-luminescence (GE Healthcare Life Science). Images were acquired and quantified using Alliance Mini HD6 system by UVITEC Ltd., Cambridge, equipped with UVI1D Software (UVITEC, 14-630,275). Detailed information for all antibodies is provided in the Supplementary Table S1.

Protein samples (2 μg) were analyzed on 12% Bis-Tris Bolt SDS-PAGE gels (ThermoFisher) with Bolt MES SDS running buffer (ThermoFisher). A Spectra Multicolor Broad Range Protein Ladder (ThermoFisher) was used as standard, and the gels were stained with Comassie Brilliant Blue (BioRad).

Histones were extracted using the Histone Extraction Kit (Abcam, Cambridge, UK). Protein concentration was quantified by DC Protein Assay (Bio-Rad, Hercules, CA, USA) and 20 µg of protein were loaded on a Bolt 10 % Bis-Tris Plus Gel, 10-Well (Thermo Fisher Scientific) using 4X Bolt LDS sample buffer and 10X Bolt Sample Reducing Agent (both Thermo Fisher Scientific). Electrophoresis war carried out at 200 V for 25–35 min using PageRuler Plus Prestained Protein Ladder, 10 to 250 kDa and Bolt MES SDS Running Buffer (both Thermo Fisher Scientific). Protein was transferred to an Immobilon-FL PVDF membrane (Merck-Millipore) at 50 V for 200 min by using Bolt Transfer Buffer (Thermo Fisher Scientific). After blocking, primary antibodies (Tri-Methyl-Histone H3 (Lys4) from Cell Signaling and Anti-Histone H3 (mono methyl K79) antibody, Anti-Histone H3 (di methyl K79) antibody, Anti-Histone H4 (symmetric di methyl R3) antibody, and Anti-Histone H3 antibody from Abcam), and the secondary antibodies (anti-rabbit IgG, IRDye 680RD, polyclonal and ant-rabbit IgG, IRDye 800CW, polyclonal from Li-Cor Biosciences) were used. Analysis was carried out at the Li-Cor Odyssey CLx using the Image Studio Software (both Li-Cor Biosciences) for documentation. Relative quantification was performed using Histone H3 as the reference protein.

Cells were lysed in the presence of cOmplete™ ULTRA Protease Inhibitor (Sigma-Aldrich, Dorset, UK, cat. 5892791001) and PhosSTOP™ Phosphatase Inhibitor (Sigma-Aldrich, cat. 4906845001), with protein quantified by Bradford detection assay using Quick Start™ Bradford Dye Reagent (BIO-RAD, Hertfordshire, UK, cat. 5000205) as previously described [48 ]. Protein extracts were prepared using NuPAGE™ LDS Sample Buffer (ThermoFisher, cat. NP0007) and NuPAGE™ Sample Reducing Agent (ThermoFisher, cat. NP0009) and electrophoresis was carried out using NuPAGE™ 4–12% Bis-Tris Gels (ThermoFisher, cat. NP0323) in Bolt™ MES SDS Running Buffer (ThermoFisher, cat. B0002) containing NuPAGE™ Antioxidant (ThermoFisher, cat. NP0005). Protein transfer was carried out in NuPAGE™ Transfer Buffer (ThermoFisher, cat. NP0006-1) onto Amersham™ Protran® Nitrocellulose Membranes (Sigma-Aldrich, cat. GE10600002). Membranes were stained with BCL2 (124) Mouse Ab (Cell Signaling Technology, cat. 15071S), MCL-1 Rabbit Ab (Cell Signaling Technology, cat. 4572S), BCL-xL (54H6) (Cell Signaling Technology, cat. 2764S), or SH-PTP2 (B-1) (Santa Cruz Biotechnology, Texas, USA, cat. sc-7384) and visualized using an Odyssey™ Fc Imaging System (LI-COR Biosciences, Cambridge, UK). Densitometry was performed using Image Studio and ratio of BCL2/MCL-1/BCL-xL:SHPTP2 calculated and normalized to parental KCL-22 cells.