The NeoPRO Femto Western ECL Substrate is a two-component chemiluminescent substrate designed for Western blotting and HRP-based protein detection. It provides a mid- to low-femtogram detection level and is particularly suitable for applications requiring very high sensitivity and reliable detection of low-abundance protein targets.

The substrate is based on a luminol-derived chemiluminescent detection system. During detection, horseradish peroxidase catalyzes the oxidation of the luminol derivative, producing light at approximately 425 nm. Enhancing components incorporated into the formulation increase the intensity and duration of the chemiluminescent signal.

The NeoPRO Femto Western ECL Substrate generates a strong chemiluminescent signal in the presence of a very low background level, providing a high signal-to-noise ratio. Its extended signal duration facilitates repeated image acquisition and optimization of exposure conditions.

The product is supplied as a two-solution kit containing a luminol derivative/enhancer solution and a peroxide solution. The two components are mixed at a 1: 1 ratio immediately before use to prepare the working solution.

• Two-component chemiluminescent substrate for Western blotting
• Designed for mid- to low-femtogram protein detection
• Very high sensitivity for low-abundance protein targets
• Strong chemiluminescent signal with a very low background level
• High signal-to-noise ratio for clear visualization of low-intensity bands
• Extended chemiluminescent signal duration
• HRP-dependent chemiluminescent detection
• Luminol derivative/enhancer and peroxide solutions supplied separately
• Working solution prepared by mixing the two components at a 1: 1 ratio
• Compatible with HRP-conjugated secondary antibodies
• Compatible with nitrocellulose and PVDF Western blot membranes
• Suitable for signal acquisition using chemiluminescence imaging systems or autoradiography film
• Recommended primary antibody dilution: 1: 5,000–1: 50,000
• Recommended secondary antibody dilution: 1: 50,000–1: 250,000
• Research use only

1. Prepare the working solution: Mix equal volumes of Solution A and Solution B at a 1: 1 ratio immediately before use.
2. Avoid cross-contamination: Use separate, clean pipette tips for each stock solution. Do not introduce Solution A into Solution B or Solution B into Solution A.
3. Prepare the membrane: Following primary and HRP-conjugated secondary antibody incubation, wash the membrane thoroughly with TBS-T buffer.
4. Rinse before detection: Remove the membrane from the washing tray, rinse it twice with TBS-T and keep it wet until application of the substrate.
5. Remove excess buffer: Allow excess buffer to drain from one corner of the membrane without permitting the membrane to dry.
6. Apply the substrate: Apply approximately 0.1 mL of prepared NeoPRO Femto working solution per cm² of membrane.
7. Incubate: Place the membrane protein-side up and ensure that the working solution completely covers the membrane surface. Incubate for approximately 1.5 minutes.
8. Acquire the signal: Detect the chemiluminescent signal using a compatible imaging device or autoradiography film.
9. Optimize exposure: When the expected signal intensity is unknown, initial exposure times of 15 seconds, 30 seconds, 1 minute and 5 minutes may be tested.
10. Optimize antibody dilutions: Adjust primary and secondary antibody concentrations when necessary to obtain a strong specific signal with minimal background.