Thermo Scientific 1-Step Ultra TMB-Blotting Solution is an enhanced single-component horseradish peroxidase (HRP) substrate for Western blotting and immunohistochemistry.
This precipitating, colorimetric Western blot substrate for HRP provides high sensitivity, increased signal-to-noise ratio and low background compared to many other chromogenic substrates. The blotting solution contains soluble TMB (3,3',5,5'-tetramethylbenzidine), which reacts very quickly with horseradish peroxidase 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 no mixing required.
- Fast – protein bands visible in less than one minute
- Fade-resistant – protein bands stable after membrane drying
- Sensitive – detection limit similar to Pierce ECL Western Blotting Substrate
- Chromogenic – no special equipment needed for visualization; produces dark blue bands
- Ready to use – no organic solvents are required to dissolve; no dilution necessary for use
- Chromogenic Western blot detection
- Chromogenic immunohistochemistry detection
- Nitrocellulose membrane blots
- PVDF membrane blots
|1-Step Ultra TMB-Blotting Solution provides sensitivity similar to Pierce ECL Western Blotting Substrate. Serial dilutions of HeLa cell lysate (7.5, 3.45, 1.88, 0.94, 0.47, 0.23 and 0.12μg) were prepared and separated by electrophoresis. The proteins were transferred to nitrocellulose membranes (Part No. 88013) and the membranes were blocked with 5% skim milk in TBS + 0.05% Tween* 20. After blocking, the membranes were incubated with Heat Shock Protein 86 Polyclonal Antibody (Hsp86) (Part No. PA3-013) at 0.5μg/mL. The membranes were washed and then incubated with 0.2μg/mL of HRP-Conjugated Goat Anti-Rabbit IgG (Part No. 31460) and then washed again. Working solution of Pierce ECL Substrate was prepared according to the instructions and added to the membrane for 5 minutes. The membrane was removed from the substrate, placed in plastic sheet protectors and exposed to CL-XPosure Film (Part No. 34090) for 30 seconds (RIGHT). Second membrane was placed in 10mL of Pierce 1-Step Ultra TMB-Blotting Solution (Part No. 37574) and the color development was stopped at 5 minutes by rinsing the membrane with water (LEFT).
|Detection with Thermo Scientific 1-Step Ultra TMB-Blotting Solution and other commercial TMB substrates. Serial dilutions of HepG2 cell lysate (15, 7.5, 3.45, 1.88, 0.94μg) were prepared and separated by electrophoresis. The proteins were transferred to nitrocellulose (TOP) membranes (Part No. 88013) and PVDF (BOTTOM) membranes (Part No. 88518). The membranes were blocked with Clear Milk Blocker 1X (Part No. 37587). After blocking, the membranes were incubated with mouse PLK-1 Monoclonal Antibody (Part No. MA1-848) and Cyclophilin B Polyclonal Antibody (Part No. PA1-027A). The membranes were washed and then incubated with 0.2μg/mL of HRP-Conjugated Goat Anti-Mouse IgG (Part No. 31430) and HRP-Conjugated Goat Anti-Rabbit IgG (Part No. 31460) and then washed again. The membranes were placed in 10mL of Pierce 1-Step Ultra TMB-Blotting Solution (Part No. 37574) (A), TMB substrate from Supplier B. The color development was stopped at 3 minutes (BOTTOM) and 5 minutes (TOP) by rinsing the membranes with ultra pure water.
Review of chromogenic Western blotting methods
Tween* 20 Detergent Solution
PVDF Transfer Membranes
Protein Molecular Weight Markers
Blocking Buffers for Protein Methods