NeoPRO Atto Western ECL Substrate

The NeoPRO Atto Western ECL Substrate is an ultra-sensitive, two-component chemiluminescent substrate designed for Western blotting and HRP-based protein detection. It provides extremely high signal intensity for the detection of trace amounts of protein and is particularly suitable for very low- and extremely 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 light output, generating very bright protein bands within short exposure times.

The NeoPRO Atto Western ECL Substrate is designed to improve the detection and interpretation of the faintest Western blot bands. Its very high sensitivity makes it suitable for experiments involving minute protein quantities, limited amounts of cell lysate or highly diluted antibodies, helping preserve valuable samples and costly primary antibodies.

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.

• Ultra-sensitive two-component chemiluminescent substrate for Western blotting
• Designed for the detection of trace amounts of protein
• Extremely high chemiluminescent signal intensity
• Suitable for extremely low-abundance protein targets
• Produces very bright bands within short exposure times
• Enhances the visualization and interpretation of faint Western blot bands
• Suitable for limited or valuable biological samples
• Supports the use of highly diluted primary and secondary antibodies
• May reduce consumption of valuable samples and expensive 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: 10,000–1: 200,000
• Recommended secondary antibody dilution: 1: 300,000–1: 1,000,000
• 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 Atto 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. Begin with a short exposure: Because NeoPRO Atto produces extremely high light emission, begin with a short exposure time and increase it only when required.
10. Optimize antibody dilutions: Dilute the antibodies sufficiently to obtain a specific signal while limiting background, saturation and rapid substrate depletion.