Introduction:

Super ECL Plus Western Blotting Substate is a chemiluminescent substrate used for chemiluminescence-based immunodetection for horse radish peroxidase (HRP) on western blotting.

• Higher antibody dilution ratios (1:2000～1:10000) can be used, which greatly saves antibodies.
• Easy to use: can be substituted for other costlier ECL Substrate without any re-optimization.
• Higher sensitivity: detect targets down to the low-picogram level.
• Longer signal duration: sustained light output for as long as 5 hours.
• More imaging options: X-ray, CCD or laser-based imagers.
• More affordable: high quality and performance at a lower price than other suppliers' similar substrates.

Application: Western blot detection for HRP tag antibody and detection of HRP-labeled probes in nucleic acid hybridization.

Storage: Store at 4℃ away from light, valid for one year. It can also be stored at room temperature for short periods of time.

Protocol (for reference only):

1. Perform western blot procedures such as SDS-PAGE, membrane transfer, blocking and antibody incubation as usual.
2. Prepare the substrate working solution as the same time of the last time membrane washing after secondary antibody incubation: Take equal volumes of solution A and solution B and mix in a clean container.

Note: It is recommended to use the working solution immediately, and it can still be used after a few hours at room temperature, but the sensitivity is slightly reduced.

3. Remove the membrane with a tweezer and lay it on the filter paper to drain washing buffer. Do not allow the membrane to dry out.
4. Immerse the membrane completely in the substrate working solution (0.125 mL working solution/cm² membrane), and make full contact with the substrate working solution. Incubate for 3 min at room temperature, and prepare for immediate exposure.

Note: Incubating for too long does not increase sensitivity but sometimes results in abnormal exposure bands. The essence of the chemiluminescence process is an enzymatic reaction. Using too little substrate working solution is not conducive to the reaction, and will also lead to uneven exposure of the bands on the membrane and significantly reduce the sensitivity. For the purpose of saving, the membrane can be cut small but do not reduce the amount of substrate working solution. 

5. Remove the membrane with a tweezer and lay it on the filter paper to drain substrate working solution. Do not wash off the substrate solution.
6. Open the X-ray film cassette, and lay a piece of plastic wrap with an area larger than the membrane on the inner surface of the cassette. Place the blot membrane in the plastic wrap and fold the plastic wrap to completely wrap the membrane. Carefully remove air bubbles and wrinkles, cut off excess plastic wrap around the edges and remove excess liquid by filter papers.
7. Place the protected membrane in a film cassette or imaging system and expose for 1 second to several minutes depending on the signal produced and image desired.

Notes:

1. It is necessary to change new tips when pipette solution A and solution B. Cross contamination will lead to the gradual failure of solution A and solution B, which will affect subsequent use effect.
2. Avoid exposure to the sun or other intense light. Prolonged exposure to strong light can result in reduced sensitivity, which can be avoided by operating in a darkroom. Short-term exposure to lab lighting is okay.
3. Always wear gloves or use clean, plastic forceps. Metallic devices (e.g., scissors) must have no visible signs of rust, which may cause speckling and/or high background.
4. Optimize your western blot procedure for best results. Variables include sample amount, gel type, transfer method, membrane type, blocking reagent, wash buffer, primary and secondary antibody concentrations, and incubation times.
5. Do not use milk in blocking or diluent buffers when using avidin/biotin detection systems. Endogenous biotin in milk can cause high background.
6. Use a sufficient volume of all solutions to ensure that membranes do not dry out.
7. Do not use sodium azide as a preservative for buffers when using an HRP detection system. Sodium azide inhibits HRP enzyme activity.
8. Please cap the solution bottle tightly to prevent failure after using.
9. Protein bands can be developed after 3 minutes incubation with substrate working buffer. The strong band luminescence can be visible to the naked eye in the darkroom, and the low-abundance protein band luminescence is weak or even invisible to the naked eye but can expose the X-ray film. Bands invisible to the naked eye can actually last for hours and sensitize X-ray film, so weak bands can be exposed for 1-10 hours. If the bands are not good after exposure, wash the membrane, re-incubate the secondary antibody, and develop again with ECL substrate. The luminescence lasts for a long time, but the fluorescence is stronger within 30 minutes after the reaction starts, and then the fluorescence will gradually weaken, so please make full use of this 30 minutes for exposure or image detection.
10. Long exposure or excess protein will deepen the background and make the band intensity change out of linearity. Underexposure results in blurred bands.
11. Since the luminescent substrate solution is extremely sensitive, it is recommended that the initial antibody concentration be 1:1000-1:4000 for the primary antibody and 1:2000-1:5000 for the secondary antibody. High antibody concentrations will result in high background or no banding, leading to failure.
12. Some plastic wraps may quench fluorescence when wrapping blot membranes, and high-quality plastic wrap should be selected.
13. Pre-stained protein markers and fluorescent-autoradiographic exposure labels allow precise determination of the location and size of bands on film.
14. Both solution A and solution B are harmful to human body, please pay attention to proper protection during operation.
15. For research use only. Not for use in diagnostic procedures.