The NeoPRO Femto PLUS Western ECL Substrate is a two-component chemiluminescent substrate designed for Western blotting and HRP-based protein detection. It provides a low-femtogram detection level and is particularly suitable for detecting very low amounts of protein and low-abundance 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 of the chemiluminescent signal.

The NeoPRO Femto PLUS Western ECL Substrate combines ultra-high sensitivity with excellent signal intensity and a wide linear dynamic range. This supports quantitative Western blot analysis while allowing highly diluted antibodies and reduced consumption of valuable reagents.

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 low-femtogram protein detection
• Ultra-high sensitivity for very low-abundance protein targets
• Excellent chemiluminescent signal intensity
• Suitable for detecting very low amounts of protein
• Wide linear dynamic range for quantitative Western blot analysis
• Supports the use of highly diluted antibodies
• May reduce consumption of valuable primary and secondary antibodies
• 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 chemiluminescence imaging systems and autoradiography film
• Recommended primary antibody dilution: 1: 5,000–1: 100,000
• Recommended secondary antibody dilution: 1: 100,000–1: 500,000
• 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 its tray, rinse it twice with TBS-T and keep it wet until the substrate is applied.
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 PLUS 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: Because of the ultra-high sensitivity of the substrate, dilute the antibodies sufficiently to obtain a specific signal while limiting background and saturation.