b'1-Step Ultra TMB-Blotting SolutionThermo Scientific 1-Step Ultra TMB-Blotting Solution is an enhanced single-component HRP substrate for western blotting. This precipitating, colorimetric western blot substrate for HRP provides high sensitivity, increased signal-to-noise ratio, and low background compared to other chromogenic substrates (Figure 37). The blotting solution contains soluble TMB (3,3,5,5-tetramethylbenzidine), which reacts very quickly with HRP enzyme to produce an insoluble dark blue precipitate that does not fade or flake. The substrate is compatible with both nitrocellulose and PVDF membranes. The blotting solution is supplied ready to use with no1-Step UltraTMB membranemixing required.TMB-Blotting Solution substrate from Supplier BA BFeatures: Fastprotein bands visible in less than one minuteNitrocellulose Fade-resistantprotein bands stable aftermembranemembrane drying Sensitivedetection limit similar to Thermo Scientific Pierce ECL Western Blotting Substrate Chromogenicno special equipment needed forC Dvisualization; produces dark blue bands Ready to useno organic solvents are required to dissolve; no dilution necessary for use PVDFmembraneFigure 37. Detection with 1-Step Ultra TMB-Blotting Solution and another commercial TMB substrate. Serial dilutions of HepG2 cell lysates (15, 7.5, 3.45, 1.88, 0.94 g) were prepared and separated by electrophoresis. The proteins were transferred to (A, B) nitrocellulose membranes and (C, D) PVDF membranes. The membranes were blocked with Pierce Clear Milk Blocking Buffer at 1X. After blocking, the membranes were incubated with Invitrogen Mouse PLK-1 Monoclonal Antibody and Rabbit Cyclophilin B Polyclonal Antibody. The membranes were washed and then incubated with 0.2 g/mL of Invitrogen Goat AntiMouse IgG (H+L) Secondary Antibody, HRP Conjugate, and Goat AntiRabbit IgG (H+L) Secondary Antibody, HRP Conjugate, and then washed again. The membranes were placed in (A, C) 10 mL of 1-Step Ultra TMB-Blotting Solution, and (B, D) TMB substrate from Supplier B. The color development was stopped at (C, D) 3 minutes and (A, B) 5 minutes by rinsing the membranes with ultrapure water.57'