ECL Plus high sensitivity illuminating liquid use method
1. Incubate and wash the membrane by conventional electrophoresis, transfer membrane, HRP labeled antibody or nucleic acid probe. Note the labeling of IgG with HRP or with the primary antibody-streptavidin-biotin-HRP sandwich method. The nucleic acid hybridization membrane was hybridized with an HRP-labeled probe and washed.
2. While preparing the HRP-labeled secondary antibody on the washing membrane, freshly prepare the luminescent working solution: take an equal volume of the solution A and B, respectively, and place it to return to room temperature, otherwise the fluorescence intensity will be weakened. It is recommended to use the working fluid immediately, and it can still be used after standing for several hours at room temperature, but the sensitivity is slightly reduced.
3. Remove the membrane with tweezers and drain the dry cleaning solution on the filter paper, but do not allow the membrane to dry completely. The membrane was completely immersed and brought into full contact with the luminescent working solution (about 0.125 ml of luminescent working solution/cm2 membrane). Immediately after incubation for 3 minutes at room temperature, the tablets were exposed. Excessive incubation times do not increase sensitivity and sometimes cause abnormal exposure bands. The essence of the luminescence process is the enzymatic reaction. The use of too little luminescent working solution is not conducive to the reaction, and it also leads to uneven exposure of the film on the film and a significant decrease in sensitivity. For the purpose of saving, the film can be cut small but the amount of luminescent liquid should not be reduced.
4. Pinch the film with tweezers and drain the working solution on the filter paper. But do not wash away the luminescent liquid.
5. Place a wrap film larger than the film on the inside of the X-ray film cassette. The hybrid film is attached to the plastic wrap, and the wrap film is folded to completely wrap the hybrid film to remove bubbles and wrinkles, and the excess wrap film at the edge portion can be cut off. Use a filter paper to remove excess luminescent working solution. The cling film covering the hybrid membrane is fixed in a cassette with a tape, and the recommended protein strip is facing upward.
6. Put X photographic film in the dark and expose it for different time, such as seconds to minutes. Development rinse.
ECL Plus Super Sensitive Liquid Note:
1. Steps 1-5 can be operated under fluorescent light; however, the sensitivity of the luminescent liquid exposed to strong light for a long time may be slightly reduced, and moving to the darkroom can be avoided. Wearing gloves can avoid leaving fingerprints on the film and keeping the film clean.
2, long exposure or protein excess, will deepen the background and make the strip strength change weak linear relationship. If the exposure is insufficient, the strip is blurred. 3. After the luminescent working solution was incubated for about 3 minutes, the strip on the film illuminates. Strong strips are visible to the naked eye in the darkroom, and low-abundance protein strips are weakly illuminated, but the naked eye is not visible but can expose X-film. The band illumination time cannot be judged by the naked eye alone. Fluorescence that is invisible to the naked eye can actually last for hours and sensitize the X film, so the weak band can be exposed for 1-10 hours. If the band is not good after exposure, wash the membrane with a membrane buffer, re-incubated the secondary antibody, and then re-illuminate and expose with ECL.
4. Due to the high sensitivity of the hypersensitive luminescent liquid, it is strongly recommended that the initial concentration of most imported antibodies be primary antibody 1:1000-1:4000, secondary antibody 1:2000-1:5000. Excessive antibody concentrations will result in high background or no bands, leading to failure! Solarbio Beijing Solarbio Science & Technology Co., Ltd /82 Http:// . Com
5. Some commercially available cling film will quench the fluorescence when it is coated with the imprinted film. High-quality cling film, such as “Klinley†brand cling film, should be selected.
6. Use the visible pre-stained protein Marker and the fluorescence-radioautoradiation exposure label to help determine the exact position and size of the strip on the film.
7. NaN3 can inhibit HRP activity. When recovering secondary antibody, NaN3 should be avoided. If it is necessary, do not exceed 0.01%.
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