Usage and precautions for ultra sensitive ECL luminescent liquid substrate (transportation and storage)
EZ ECL Pico chemiluminescence solution is a highly sensitive enhanced chemiluminescence (ECL) HRP substrate that can help users achieve protein detection below picogram level during immunoblotting analysis. The characteristics of EZ ECL Pico substrate include:
High sensitivity: detecting protein bands with low Pic level on nitrocellulose or PVDF membranes.
Long signal duration: Under optimized conditions, the imprinted bands incubated with substrates can continuously output detectable light signals for 6-8 hours.
• Reagent stability: The reagent kit can be stored stably at room temperature for up to 6 months, and at 4 ℃ for 1 year without affecting its use.
Suggested dilution concentration:
• Primary antibody concentration: It is recommended to start with 1mg/mL stock solution at a ratio of 1:1000-1:5000 or 0.2-1ug/mL
Secondary antibody concentration: It is recommended to start with 1mg/mL stock solution at a concentration of 1:20000-1:100000 or 10-50ng/mL
Product components
| 组分名称 | A液 | B液 |
| AP34L024（100 mL） | 50 mL | 50 mL |
| AP34L025（500 mL） | 250 mL | 250 mL |
Transportation and storage
Blue ice transportation. Store at 4 ℃ away from light, with a shelf life of 12 months.
usage method
Remove the imprinting membrane, add a suitable blocking solution and incubate in a greenhouse for 20-60 minutes while shaking to block non-specific protein binding sites on the membrane.
Remove the membrane from the sealing solution and incubate it with the primary antibody working solution in a greenhouse for 1 hour while shaking; Or incubate overnight at 28 ℃ without shaking.
Add sufficient washing buffer onto the membrane to ensure that the buffer covers the membrane. Shake and incubate for ≥ 5 minutes, replace the washing buffer and repeat this step 4-6 times. Increasing the volume of washing buffer, washing frequency, and washing time can help reduce background signals. 【 Note 】: ① Before incubation, brief rinsing of the membrane in washing buffer can improve washing efficiency. ② The dilution of HRP labeled secondary antibody is very important as suggested in the previous text。
Incubate the HRP labeled secondary antibody working solution with the membrane in a greenhouse for 1 hour while shaking。
Repeat step 3 to remove unbound HRP labeled secondary antibodies. [Note]: After incubation with HRP labeled secondary antibody, the membrane must be washed.
Mix solution A and solution B in equal proportions to prepare the working solution. 0.01~0.1 mL of working solution is used per cm2 of membrane, and the working solution can be stable for 8 hours in a greenhouse。
[Note]: Exposure to sunlight or other strong light may damage the working fluid. For best results, please store this working fluid in an amber bottle; The common lighting in the laboratory will not damage the working fluid。
Incubate the imprinting membrane in the working solution for 5 minutes. Remove the imprint film from the working fluid and place it in a plastic sheet or clean plastic paper (film). Use a piece of absorbent paper to remove excess liquid and carefully press out bubbles between the imprint and the plastic paper.
Place the imprint film wrapped in plastic paper (film) in the film cassette, with the protein side facing up, and turn off all lights except for those suitable for film exposure (such as the red safety light). [Note]: ① The film must be kept dry during the exposure period; ② Ensure that excess substrates are removed from the membrane and plastic paper *; ③ Use gloves throughout the entire film processing period.
Place the X-ray film on top of the film. Suggest a first exposure of 60 seconds. Afterwards, the exposure time can be adjusted to achieve the best results. The chemiluminescence reaction is strong during the first 5-30 minutes after substrate incubation. This reaction can last for several hours, but the intensity will decrease over time. If the substrate is incubated for a longer period of time and then exposed, the exposure time may need to be extended to obtain a stronger signal. If phosphorescent storage imaging devices (such as Bio Rad's molecular imaging system) or CCD cameras are used, longer exposure times may be required. [Note]: Any movement between films may create artificial non-specific signals on the film.
Develop the film using appropriate developer and fixative. If the signal is too strong, shorten the exposure time or peel off the imprint film and reduce the antibody concentration for retesting.
matters needing attention
This product is only for scientific experimental research and should not be used in clinical diagnosis, treatment, or other fields.
To achieve optimal results, it is necessary to optimize all components of the system, including sample size, concentrations of primary and secondary antibodies, and types of membranes and blocking reagents.
The use of this product requires a lower antibody concentration than the use of precipitation colorimetric HRP substrate for detection. To optimize antibody concentration, please perform a systematic dot blot analysis。
There is no single blocking reagent that is optimal for all systems, so it is essential to find the most suitable blocking buffer for each immunoblotting detection system. Blocking reagents may cause cross reactivity with antibodies, leading to non-specific signals. Blocking buffer solution can also affect the sensitivity of the system. When switching from one substrate to another, signal attenuation or background increase may sometimes occur, possibly due to the unsuitability of the blocking buffer for the new detection system.
When using affinity/biotin detection systems, avoid using milk as a blocking reagent because milk contains an indefinite amount of endogenous biotin, which can lead to high background signals。
Ensure the use volume of washing buffer, blocking buffer, antibody solution, and substrate working solution to ensure that the imprinting membrane * is covered by liquid throughout the entire experimental process and to avoid membrane drying. Increasing the usage of blocking buffer and washing buffer can reduce non-specific signals.
For optimal results, please use a shaker during the incubation process.
Add Tween20 (final concentration 0.05~0.1%) to the blocking buffer and diluted antibody solution to reduce non-specific signals. Use high-quality products such as stain removers. It is stored in ampoules with low levels of peroxides and other impurities.
Do not use diedanna as a preservative for buffer solutions. Diedanna is an inhibitor of HRP.
Avoid direct contact between hands and the membrane. During the experiment, gloves should be worn or clean tweezers should be used。
All equipment must be clean and free from foreign substances. Metal instruments (such as scissors) must not have visible rust stains. Rust may cause spot formation and high background。
The substrate working solution can be stable for 8 hours at room temperature. Sunlight or any other strong light may damage the substrate. To achieve optimal results, store the substrate working solution in an amber bottle and avoid long-term exposure to any strong light. Short term exposure to laboratory lighting will not damage the working solution.
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