NeoPRO Pico PLUS Western ECL Substrate

The NeoPRO Pico PLUS Western ECL Substrate is a two-component chemiluminescent substrate developed for Western blotting and HRP-based protein detection. It provides a mid-femtogram detection level and combines high sensitivity with a broad linear dynamic range for the detection and quantification of both low- and high-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, generating light at approximately 425 nm. Enhancing components incorporated into the formulation increase the intensity and duration of the chemiluminescent signal.

The NeoPRO Pico PLUS Western ECL Substrate is supplied as a two-solution kit containing a luminol derivative/enhancer solution and a peroxide solution. The two solutions are mixed at a 1: 1 ratio immediately before use to prepare the working solution.

Its high sensitivity, broad linear dynamic range and extremely long signal duration make it a versatile substrate for Western blot experiments requiring reliable detection of proteins with different abundance levels. The formulation is designed to require less optimization while supporting accurate chemiluminescent signal acquisition using imaging systems or autoradiography film.

• Two-component chemiluminescent substrate for Western blotting
• Designed for mid-femtogram protein detection
• High sensitivity for detecting low-abundance protein targets
• Broad linear dynamic range for analysis of both low- and high-abundance proteins
• Suitable for accurate quantitative Western blot analysis
• Extremely long chemiluminescent signal duration
• Versatile formulation requiring less assay optimization
• 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 chemiluminescent imaging systems and autoradiography film
• Recommended primary antibody dilution: 1: 1,000–1: 15,000
• Recommended secondary antibody dilution: 1: 25,000–1: 150,000
• 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. Prepare only the volume required for the membrane.
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 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 Pico 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: Adjust primary and secondary antibody concentrations when necessary to obtain a strong specific signal with minimal background.