Criterion tgx stain free precast gel

The molecular weight distribution was analyzed using SDS-PAGE under non-reducing and reducing conditions according to the method used by Laemmli [30 (link)], with slight modifications. Briefly, 5 μg/μL protein solution (based on dry matter) was prepared in 1× treatment buffer (50% (v/v) 2× Tris-HCl treatment buffer, 50% (v/v) phosphate buffer (pH 7)). The 2× treatment buffer was prepared using 0.125M from the 4× stacking gel buffer (0.5M Tris, adjusted with HCl to pH 6.8), 4% from 10% SDS, 20% glycerol, and 0.02% Bromophenol Blue, while for reduction conditions 0.2M dithiothreitol was added. The samples were heated (95 °C, 5 min) prior to centrifugation at 12,045× g for 3 min (MiniSpin, Eppendorf AG, Hamburg, Germany). The supernatants were mixed 1:10 (v/v) with 1× treatment buffer, from which 3 μL was added into the gel pocket of the Bio-Rad 4–20% Criterion™ TGX Stain-Free™ Precast Gels. The Broad Range™ Unstained Protein Standard was used as the molecular weight marker. The running time was 30 min, followed by staining using Coomassie Brilliant Blue R-250. Finally, gel images were obtained using an EZ Imager (Gel Doc™ EZ Imager—Bio-Rad). Protein bands and their intensities were calculated using Image Lab Software. SDS-PAGE was performed in duplicate, with each sample being prepared two times independently.

Immunoprecipitation of KaiB‐FLAG and associated protein complexes in a clock reaction (Fig 4A) was done as previously described (Phong et al, 2013) using monoclonal anti‐FLAG M2 antibody (Sigma‐Aldrich, product number F1804), and elution with 3xFLAG peptide. The supernatant was analyzed by SDS–PAGE on 4–20% Criterion TGX Stain‐Free Precast Gels (Bio‐Rad) and stained with SYPRO Ruby (Bio‐Rad). 1.5 μM GFP was added to the reaction mixture at the beginning of the time course and served as an internal standard to correct for changes in protein concentration due to handling. The relative supernatant KaiA concentration was determined as a ratio of KaiA band intensity in each lane to the GFP band intensity and is normalized as percentage of the largest ratio in the time course (Fig EV3D).

Standard Radioimmunoprecipitation assay (RIPA) buffer was used to harvest protein lysates from tumour tissues while a Bradford assay was performed to determine protein concentration. Protein samples (50 μg), prepared with Laemmli’s sample buffer, were loaded onto 4–20% Criterion™ TGX Stain-Free™ Precast Gels (BioRad, CA, USA). Proteins were separated at 100 V for 10 min and 120 V for 60 min in Tris/Glycine/SDS running buffer (BioRad, CA, USA). Proteins were transferred onto Polyvinylidene difluoride (PVDF) membranes (Trans-Blot® Turbo RTA Midi PVDF transfer kit, BioRad, CA, USA) with the Trans-Blot® Turbo Transfer System (BioRad, CA, USA) with the conditions of 25 V, 1.0A, 30 min. The Stain-Free™ properties of the membranes were utilized on the Chemidoc™ MP System (BioRad, CA, USA) to determine the total protein intensities of each membrane. Membranes were blocked in 5% milk for 1 h and incubated in primary antibody, prepared in tris-buffered saline with tween 20 (TBS-T), at 4 °C overnight. On the following day membranes were incubated in secondary antibody, prepared in TBS-T, for 1 h at RT. Antibody details are listed in the Additional file 1. After incubation, membranes were developed on the Chemidoc™ MP System with Clarity™ Electrochemiluminescence (ECL) Substrate (BioRad, CA, USA).

The different field pea seeds (Pisum sativum L.) were kindly provided by Norddeutsche Pflanzenzucht Hans-Georg-Lembke KG (Holtsee, Germany) and are shown in Table 1. The Broad Range™ Unstained Standard, 4–20% Criterion™ TGX Stain-Free™ Precast Gels, and Coomassie blue R-250 were purchased from Bio-Rad Laboratories GmbH (Feldkirchen, Germany). Sodium dihydrogen phosphate, sodium dodecyl sulfate, and sodium monohydrogen phosphate were purchased from Sigma-Aldrich Chemie GmbH (Munich, Germany). All chemicals used in this study were of analytical grade.

The molecular weight distribution was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli [39 (link)] with slight modifications and as described in detail in García Arteaga, Apéstegui Guardia [18 (link)]. Briefly, 5 μg/μL protein solution (based on dry matter) was prepared in 1× reducing buffer (50% (v/v) 2× Tris-HCl reducing buffer, 50% (v/v) phosphate buffer (pH 7)). The samples were heated (95 °C, 5 min) prior to centrifugation at 12,045× g for 3 min (MiniSpin, Eppendorf AG, Hamburg, Germany). An aliquot of 3 μL of the supernatants was added into the gel pocket of the Bio-Rad 4–20% Criterion™ TGX Stain-Free™ Precast Gels. The Broad Range™ Unstained Protein Standard was used as the molecular weight marker. The running time was 30 min, followed by staining using Coomassie Brilliant Blue R-250. Finally, gel images were obtained using an EZ Imager (Gel Doc™ EZ Imager, Bio-Rad Laboratories, Feldkirchen, Germany). SDS-PAGE was performed in duplicate, with each sample being prepared two times independently.