Instructions for Use and Precautions Regarding the Ultra-Sensitive ECL Luminescent Substrate (Transportation and Storage)

EZ ECL pico chemiluminescent solution is a highly sensitive, enhanced chemiluminescence (ECL) HRP substrate that enables users to detect proteins at sub-piconewton levels in Western blotting assays. (Key features of the EZ ECL pico substrate:
• High sensitivity: detects protein bands at the picogram level on nitrocellulose or PVDF membranes.
• Signal duration: Under optimized conditions, the blot bands incubated with the substrate can sustain a detectable luminescent signal for 6–8 hours.
• Reagent stability: The kit remains stable at room temperature for up to 6 months, and can be stored at 4°C for 1 year without compromising performance.
Recommended dilution concentration:
• Primary antibody concentration: It is recommended to start with a 1 mg/mL stock solution at a dilution of 1:1,000 to 1:5,000, or 0.2–1 µg/mL.
• Secondary antibody concentration: It is recommended to prepare a 1 mg/mL stock solution and dilute it to 1:20,000–1:100,000 or 10–50 ng/mL.

Product ingredients

Transportation and Storage
Blue ice transport. Store at 4°C away from light; shelf life: 12 months.
Instructions for Use

1. Remove the membrane, add an appropriate blocking solution, and incubate at room temperature for 20–60 minutes with gentle shaking to block non-specific protein-binding sites on the membrane.

2. Remove the membrane from the blocking solution and incubate it with the primary antibody working solution at room temperature for 1 hour with gentle shaking, or incubate overnight at 28°C without shaking.

3. Add an adequate volume of wash buffer to the membrane, ensuring that the buffer fully covers the membrane. Incubate with shaking for at least 5 minutes, then replace the wash buffer and repeat this step 4–6 times. Increasing the volume of wash buffer, the number of washes, and the duration of each wash can help reduce background signal.
   [Note]: ① Prior to incubation, a brief rinse of the membrane in wash buffer can enhance washing efficiency. ② As recommended above, using the HRP‑conjugated secondary antibody at the suggested dilution is crucial.

4. Incubate the HRP‑labeled secondary antibody working solution with the membrane at room temperature for 1 hour, with gentle shaking.

5. Repeat step 3 to remove unbound HRP‑conjugated secondary antibody. [Note]: Following incubation of the membrane with the HRP‑conjugated secondary antibody, thorough washing is essential.

6. Mix solution A and solution B in equal proportions to prepare the working solution. Use 0.01–0.1 mL of the working solution per cm² of membrane; the working solution remains stable for 8 hours at room temperature.
   [Note]: Exposure to sunlight or other intense light may degrade the working solution. For optimal results, store this solution in an amber‑colored bottle; typical laboratory lighting will not adversely affect the solution.

7. Incubate the blotting membrane in the working solution for 5 minutes. Remove the membrane from the working solution and place it on a plastic sheet or a clean plastic film; use absorbent paper to blot away excess liquid, and carefully press out any air bubbles trapped between the blot and the plastic film.

8. Place the imprint film, wrapped in plastic wrap (film), into the film cassette with the protein side facing up, and turn off all lights except for those suitable for exposing the film, such as a red safety light.
   [Note]: ① The film must be kept dry during exposure; ② Ensure that any excess substrate is removed from the membrane and plastic sheet; ③ Wear gloves throughout the entire film‑processing procedure.

9. Place the X-ray film on top of the membrane. A 60‑second exposure is recommended for the first run; subsequent exposures can be adjusted to optimize results. The chemiluminescent signal is most intense during the first 5–30 minutes after substrate incubation. This signal may persist for several hours, but its intensity diminishes over time; if exposure occurs long after substrate incubation, the exposure time may need to be extended to obtain a stronger signal. When using phosphorimaging systems (such as Bio-Rad’s Molecular Imager) or CCD cameras, longer exposure times may be required.
   [Note]: Any movement between the film and the membrane may introduce artificial, non-specific signals on the film.

10. Develop the film using appropriate developer and fixer solutions. If the signal is too strong, shorten the exposure time, or strip the membrane and repeat the assay at a lower antibody concentration.

Precautions

1. This product is intended solely for scientific research and must not be used for clinical diagnosis, treatment, or any other purposes.

2. To achieve optimal results, all components of the system must be optimized, including sample volume, concentrations of primary and secondary antibodies, and the type of membrane and blocking reagent.

3. Using this product requires a lower antibody concentration than that needed when detecting with the precipitation‑based HRP substrate. To optimize the antibody concentration, perform a systematic dot‑blot analysis.

4. No single blocking reagent is optimal for all systems; therefore, it is essential to identify the most suitable blocking buffer for each Western blotting system. Blocking agents may cross‑react with antibodies, leading to non‑specific signals. Moreover, the choice of blocking buffer can influence the assay’s sensitivity. When switching from one substrate to another, signal attenuation or increased background may occur, often because the original blocking buffer is not well suited to the new detection system.

5. When using the avidin–biotin detection system, avoid using milk as a blocking reagent, as milk contains variable amounts of endogenous biotin, which can lead to high background signals.

6. Ensure adequate volumes of wash buffer, blocking buffer, antibody solution, and substrate working solution to maintain complete liquid coverage of the blot membrane throughout the experiment, thereby preventing the membrane from drying. Increasing the volumes of blocking buffer and wash buffer can help reduce non-specific signals.

7. For optimal results, please use a shaker during the incubation step.

8. Add Tween 20 (final concentration 0.05–0.1%) to the blocking buffer and the diluted antibody solution to reduce non-specific signals. Use high-quality reagents, such as detergents. The product is supplied in ampoules with very low levels of peroxides and other impurities.

9. Do not use diedanna as a preservative in the buffer. Diedanna inhibits HRP.

10. Avoid direct contact between your hands and the membrane; wear gloves or use clean forceps throughout the experiment.

11. All equipment must be clean and free of foreign matter. Metal instruments, such as scissors, must not exhibit visible rust. Rust can lead to spot formation and a high background signal.

12. The substrate working solution is stable for 8 hours at room temperature. Exposure to sunlight or any other intense light may degrade the substrate; to achieve optimal results, store the substrate working solution in an amber‑colored vial and avoid prolonged exposure to strong light. Brief exposure to standard laboratory lighting will not adversely affect the solution.