NeoPRO Pico Western ECL Substrate

The NeoPRO Pico Western ECL Substrate is a two-component chemiluminescent substrate developed for Western blotting and HRP-based protein detection. It provides a low-picogram detection level and is particularly suitable for detecting abundant protein targets in routine Western blot experiments.

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 Pico Western ECL Substrate 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 application to the Western blot membrane.

Its stable light output and low-picogram sensitivity make it suitable for routine analysis of highly expressed proteins. The substrate can be used with standard Western blot workflows involving SDS-PAGE, membrane transfer, blocking, primary antibody incubation and HRP-conjugated secondary antibody detection.

• Two-component chemiluminescent substrate for Western blotting
• Designed for low-picogram protein detection
• Suitable for abundant protein targets and samples
• Provides stable chemiluminescent light output
• 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
• Suitable for routine Western blot applications
• 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: 500–1: 5,000
• Recommended secondary antibody dilution: 1: 20,000–1: 100,000
• 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 vice versa.
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 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.