The UltraScence Femto Plus Western Substrate, as a luminol-based enhanced chemiluminescent substrate, is sensitive and compatible with conducting immunoblots with horseradish peroxidase (HRP) – conjugated secondary antibodies. The UltraScence Femto Plus Western Substrate is designed for the detection of the target proteins in amounts that are too small to be seen with typical ECL substrates. The mid femtogram to low femtogram detection of antigen is enabled by UltraScence Femto Plus Western Substrate’s excellent sensitivity and long signal duration. Further, its long chemiluminescent signal duration makes both digital and film-based imaging possible without any loss of the signal. Appropriate primary and secondary antibody dilutions are suggested for attaining optimal signal intensity and duration. 

Product Name: UltraScence Femto Plus Western Substrate

Product Item No.: CCH375-B100ML

Size: 100ml

Substrate Type: HRP (Horseradish Peroxidase) Substrate 

Contents/Storage: Store at 4℃ for 2 years 

US Patent No.: US10,711,185

- No optimization required.
Switching to the UltraScence Femto Plus Western Substrate from other brands, such as Thermo Scientific™ SuperSignal™ West Femto, does not require optimization or protocol changes.

- High degree of sensitivity and enhanced chemiluminescence duration.
UltraScence Femto Plus Western Substrate enables an accurate mid femtogram to low 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.

Figure 1. Membranes were probed with GFP tag Rabbit PolyAb (Cat No. 50430-2-AP, 1:10,000) and then with Goat Anti-rabbit IgG/HRP secondary antibody (Cat No. SA00001-2, 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 400 μL of UltraScence Femto Plus Western substrate (CCH375). The blots were simultaneously exposed for 5seconds, 10 seconds, 30 seconds, 60 seconds and 180 seconds and using Chemlux SPX-600 Series digital imaging system.

* ImmobilonTM Western Chemiluminescent HRP Substrate is a seperatiliy registered trademark of Merck Miilipore. SuperSignal West Femto is a registered trademark of Thermo Fisher Scientific. The trademark holder is not affiliated with Bio-HeliX Co., Ltd. and does not recognize this product.

Figure 2. The membrane images show that UltraScence Femto Plus Western Substrate provide sensitivity the same as Thermo ScientificTM SuperSignalTM West Femto. They can both clearly detect 5 ng EGFP after exposure for 180 sec. Membranes were probed with GFP tag Rabbit PolyAb (Cat No. 50430-2-AP, 1:10,000) and then with Goat Anti-rabbit IgG/HRP secondary antibody (Cat No. SA00001-2, 1:10,000) after serial dilution EGFP (Enhanced Green Fluorescent Protein) was prepared and applied in electrophoresis and protein transfer. Identical blots were incubated with 400 μL of UltraScence Femto Plus Western substrate (CCH375-B100ML). The blots were simultaneously exposed for 5, 10, 30, 60, and 180 seconds using Chemlux SPX-600 Series digital imaging system.

* SuperSignal West Femto is a registered trademark of Thermo Fisher Scientific. The trademark holder is not affiliated with Bio-HeliX Co., Ltd. and does not recognize this product.

Figure 3. 24-hour stability test of UltraScence Femto Plus Western Substrate

The Luminol and Peroxide solution of UltraScence Femto Plus were mixed in a 1:1 ratio and having 400 uL treated on each blot after taken out from 4℃ for 1hr, 12 hrs and 24 hrs. The blots were simultaneously exposed for 5, 10, 30, 60, and 180 seconds using Chemlux SPX-600 Series digital imaging system. The EGFP (Enhanced Green Fluorescent Protein) was serially diluted for electrophoresis, after the proteins were transferred to the membrane, they were blocked and probed with GFP tag Rabbit PolyAb (Cat No. 50430-2-AP, 1:10,000), and followed by the Goat Anti-rabbit IgG/HRP secondary antibody (Cat No. SA00001-2, 1:10,000).

Figure 4. 4-hour signal duration with UltraScence Femto Plus Western Substrate

The EGFP (Enhanced Green Fluorescent Protein) was serially diluted for electrophoresis, after the proteins were transferred to the membrane, they were blocked and probed with GFP tag Rabbit PolyAb (Cat No. 50430-2-AP, 1:10,000), and followed by the Goat Anti-rabbit IgG/HRP secondary antibody (Cat No. SA00001-2, 1:10,000). Immerse the membrane in a mixed luminol and peroxide solution and check the signal at different times. The blots were simultaneously exposed for 5, 10, 30, 60, and 180 seconds using the Chemlux SPX-600 Series digital imaging system.

Q: From Bio-Helix’s UltraScence ECL substrates portfolio, which level of ECL fits best my Western blot applications?
A: UltraScence ECL substrates series is compatible with the use from mid femtogram to low femtogram level detections. Please kindly refer to the ECL selection guide of UltraScence Western substrate as the below table:

(a) CCH321-B100ML, UltraScence Pico Plus Western Substrate

(b) CCH345-B100ML, UltraScence Pico Ultra Western Substrate

(c) CCH375-B100ML, UltraScence Femto Plus ECL Western Substrate

(d) CCH385-B100ML, UltraScence Atto ECL Western Substrat