EZ ECL flightChemiluminescent liquid (Flygram grade)

Product Description

EZ ECL FlygChemiluminescent liquid is a highly sensitive enhanced chemiluminescence solution currently available(ECL)HRPSubstrate can help users achieve low titer protein detection during immunoblotting analysis(<1×10^-13The-14g)Extremely saves antibodies.West FemoThe substrate can be horseradish peroxidase(HRP)The immunoblotting experiment of the conjugate provides bright signals and Fick level detection sensitivity. thatECLThe substrate is compatible with various membranes, blocking solutions, and wide range antibody dilutions, meeting the immunoblotting application needs of users with excellent performance, versatility, and high cost-effectiveness.

West FemoCharacteristics of substrates：

•high sensitivityDetecting nitrocellulose membrane orPVDFThe protein bands on the membrane at the Fick level.

•Long signal durationUnder optimized conditions, the imprinted bands incubated with substrates can continue to output6~8 hThe detectable optical signal.

Suggested dilution concentration：

•Primary antibody concentrationSuggestion:1 µg/mLDilution of storage solution1:5,000to1:100,000times

•Secondary antibody concentrationSuggestion:1 µg/mLDilution of storage solution1:100,000to1:500,000times

Order Information

Product components

Transportation and storage

Blue ice transportation.4Store in a dark and dry place at ℃, expiration date12A month.

Usage

1. Remove the imprint film, add a suitable sealing solution, and incubate in a greenhouse20~60 minSimultaneously oscillate to block non-specific protein binding sites on the membrane.

2. Remove the membrane from the sealing solution and incubate it with the primary antibody working solution in a greenhouse1 hSimultaneously oscillating; or28Incubate overnight at ℃ without shaking.

3. Add sufficient washing buffer onto the membrane to ensure that the buffer completely covers the membrane. Oscillatory incubation ≥5 minReplace the washing buffer and repeat this step4~6Next time. 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 will improve washing efficiency. ② As suggested earlierHRPIt is very important to mark the dilution of secondary antibodies.

4. Translate into EnglishHRPIncubate the labeled secondary antibody working solution with the membrane in a greenhouse1 hSimultaneously oscillating.

5. Repeat steps3To remove unbound onesHRPMark the secondary antibody.[Note]:Membrane andHRPAfter incubation with the secondary antibody, it must be thoroughly washed.

6. Translate into EnglishASolution andBMix the liquid in equal proportions to prepare the working fluid. eachcm²Membrane usage0.01~0.1 mLWorking fluid, working fluid can be stable in a greenhouse8 h.[Note]:Exposure to sunlight or other strong light may damage the working fluid. To achieve optimal results, please store this working fluid in an amber bottle; The common lighting in the laboratory will not damage the working fluid.

7. Incubate the imprint membrane in the working solution5 minRemove 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.

8. 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 completely removed from the membrane and plastic paper; ③ Use gloves throughout the entire film processing period.

9. Translate into EnglishXPlace the optical film on top of the film. Suggestion * One exposure60 sAfterwards, the exposure time can be adjusted to achieve the best results. Chemiluminescence reaction before substrate incubation5~30 minDuring this period, it was extremely intense. 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 using phosphorescent storage imaging devices (such asBio-RadMolecular imaging system) orCCDThe camera may require a longer exposure time.[Note]:Any movement between films may create artificial non-specific signals on the film.

10. 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.

Precautions

1. This product is intended for scientific experimental research and must not be used in clinical diagnosis, treatment, or other fields.

2. 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.

3. Using this product is more effective than using precipitation colorimetryHRPThe antibody concentration required for substrate detection is low. To optimize antibody concentration, please perform a systematic dot blot analysis.

4. 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.

5. Using avidin/When using a biotin detection system, avoid using milk as a blocking reagent because milk contains an indefinite amount of endogenous biotin, which can lead to high background signals.

6. Ensure the use volume of washing buffer, blocking buffer, antibody solution, and substrate working solution to ensure that the imprinting membrane is completely covered by the 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.

7. To achieve optimal results, please use a shaker during the incubation process.

8. Translate into EnglishTween20(final concentration0.05~0.1%)Add 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.

9. Do not use sodium azide as a preservative for buffer solutions. Sodium azide isHRPThe inhibition.

10. Avoid direct contact between hands and the membrane, and wear gloves or use clean tweezers during the experiment.

11. 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.

12. The substrate working solution is stable at room temperature8 hSunlight 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.

Related product recommendations

EZ TransCell transfection reagent(Efficient) (Item Number:AC04L092）

EZ DNA MarkerSeries（ Item No.AN33L086）