Odyssey one color protein molecular weight marker

Protein was harvested using lysis buffer (10 mM Tris pH 7.5, 150 mM NaCl, 10 mM KCl, 0.5% deoxycholate, 0.5% Tween, 0.5% IGePawl, 0.1% SDS). Protein content was quantified using DC Protein Assay (BioRad; Hercules, CA, USA). 50–80 μg of protein were loaded into gel (Bis-Tris 4–12%, 1.5 mm) with 4x loading buffer (200 mM Tris pH 6.8, 40% glycerol, 8% SDS, 0.1% Bromophenol Blue), and 5% β-mercaptoethanol. Gel was run using MES running buffer (Tris Base 50 mM pH 7.3, MES 50 mM, SDS 0.1%, EDTA 1 mM). Novex Sharp Pre-Stained Protein Standard, SeeBlue Plus2 Pre-stained Protein Standard (Thermo Fisher), or Odyssey One-Color Protein Molecular Weight Marker (LI-COR) were used as molecular markers. Gel was transferred using Bis-Tris transfer buffer (25 mM bicine, 25 mM bis-tris, 1 mM EDTA, 10% methanol). Blots were blocked for 1 h in TBST (24.8 mM tris acid & base, 1.5 NaCl, 0.5% Tween 20) with 5% milk. Primary antibodies were used 1:1000 or 1:500 with HRP-conjugated chemiluminescent secondary antibodies 1:1000 in TBST with 5% BSA (primary) or milk (secondary). Blots were imaged using Clarity Max Western ECL Substrate (BioRad) using ChemDoc MP Imaging System (BioRad).