UltraScence Atto Western Substrate

The UltraScence Atto Western Substrate, as a luminol-based enhanced chemiluminescent substrate, is the most sensitive and brightest ECL Western Substrate among our UltraScence product lines for low-femtogram to high-attogram detection of antigen with excellent sensitivity and long signal duration. UltraScence Atto Western Substrate is compatible with conducting immunoblots with horseradish peroxidase (HRP) – conjugated secondary antibodies. Further, its long chemiluminescent signal duration makes both digital and film-based imaging possible without any loss of the signal.

• No optimization required. Switching to the UltraScence Pico Plus Western ECL Substrate from other brands, such as Pierce ECL and GE Healthcare, does not require optimization or protocol changes.
• High degree of sensitivity and enhanced chemiluminescence duration. UltraScence Pico Plus Western ECL Substrate enables an accurate low picogram to high femtogram detection of protein on the same immunoblot after a single exposure.
• Optimized for use with PVDF and nitrocellulose membranes.
• Compatible with Western Blotting Markers.
• Optimized for film- and CCD-based imaging.

1. Keep the membrane moist in the wash buffer while preparing the substrate mixture. Please ensure the membrane does not dry out during the subsequent steps.
2. Mix Luminol solution and Peroxide Solution in a 1:1 ratio, and thoroughly agitate the chemiluminescent substrate solution well for preparing the 0.1 ml of solution / cm² of membrane.
- For a mini-sized membrane (7 x 8.5 cm), 4 ml of solution is sufficient.
- For a midi-sized membrane (8.5 x 13.5 cm), 10 ml of solution is sufficient.
3. Place the membrane with the protein side up on a clear and level surface or in a clean container.
4. Remove the membrane from the chemiluminescent substrate solution and drain off excessive solution.
5. Place the membrane in a plastic sheet protector or in plastic wrap to prevent the membrane from drying.
6. Image the membrane with a digital imager or by exposing to the X-ray film.

Membranes were probed with GFP tag Rabbit PolyAb diluted at 1:10,000 of and then with Goat Anti-rabbit IgG/HRP secondary antibody (1:10,000) after serial dilution EGFP (Enhanced Green Fluorescent Protein) were prepared and applied in electrophoresis and protein transfer. Identical blots were incubated with the Western substrate. The blots were simultaneously exposed for 5 seconds, 10 seconds, 30 seconds, 60 seconds, and 180 seconds using ChemluxSPX-600 Series digital imaging system. The comparison data is shown below.

• A compatible Chemiluminescence or X-ray Imaging Systems.
• A plastic sheet protector or plastic wrap to prevent the membrane from drying.