The UltraScence Femto Plus Western Substrate Powder, as a luminol-based enhanced chemiluminescent substrate, is sensitive and compatible with conducting immunoblots with horseradish peroxidase (HRP) – conjugated secondary antibodies. The low picogram to low femtogram detection (mid-atto) 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 Powder
Product Item No.: CCH375-P10L
Sizes: 5L x 2 / Set
Substrate Type: HRP (Horseradish Peroxidase) Substrate
Contents/Storage: Upon receipt make the ECL powder form into liquid form. It could be stable for up to 2 years at 4°C.

-　Ready-to-ship and significantly longer shelf life in the powder form!

-　Significant reductions in transportation and storage costs, resulting in less carbon footprint!

-　Empower custom production anywhere in the world!

-　Significantly more sensitive than leading manufacturers on the market.

Figure 1. 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 400 μL of UltraScence Femto Plus Western substrate (CCH375). The blots were simultaneously exposed for 5 seconds, 10 seconds, 30 seconds, 60 seconds and 180 seconds and using Chemlux SPX-600 Series digital imaging system.

Figure 2. 24-hour signal duration with 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 diluted solution (1:10,000), and followed by the Goat Anti-rabbit IgG/HRP secondary antibody (1:10,000).

Q: What’s the shelf life of UltraScence Pico Plus/Pico Ultra/Femto/Femto Plus Western Substrate in liquid form?
A: 2 years at 4°C.

Q: What’s the shelf life of UltraScence Pico Plus/Pico Ultra/Femto/Femto Plus Western Substrate in powder form?
A: Before you dissolve the powder ECL into liquid form, the shelf life could be more than 2 years in appropriate storage conditions. After you dissolve the powder ECL into liquid form, the shelf life of the liquid ECL: Store at RT 4°C for 2 years.

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 low picogram 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) CCH365-B100ML, UltraScence Femto ECL Western Substrate
(d) CCH375-B100ML, UltraScence Femto Plus ECL Western Substrate

(e) CCH385-B100ML, UltraScence Atto Western Substrate

Q: What’s the volume and weight for the powder ECL substrates, CCH375-P10L?
A: Our powder ECL substrates, CCH375-P10L, (~200g per package) could be dissolved to be 10L (=5L+5L) Western Substrate solutions.

》Powder dissolution

1. Prepare two 5L bottles. One amber bottle is for luminol solution, and other normal bottle is for peroxide solution. (Make sure the bottles are acid and alkali resistant.)
2. Unpack the bag and pour all the powder in bottle.
3. Rinse residual powder in bag with 10% (v/v) ethanol solution.
4. Add 10% (v/v) ethanol solution to 5L.

》Reaction Setup

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.

1. Place the membrane with the protein side up on a clear and level surface or in a clean container.
2. Remove the membrane from the chemiluminescent substrate solution and drain off excessive solution.
3. Place the membrane in a plastic sheet protector or in plastic wrap to prevent the membrane from drying.
4. Image the membrane with a digital imager or by exposing to the X-ray film.

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