West Pico ECL Substrate

Product Introduction:

West Pico hypersensitive luminescent solution is used to detect antibodies and associated antigens that directly or indirectly label HRP. Western Blot for HRP labeled antibodies and nucleic acid hybridization for HRP labeled probes. Due to the unique luminescent substrate system, West Pico supersensitive luminescent solution is the most sensitive commercial fluorescent ECL detection reagent with extremely high sensitivity and high signal-to-noise ratio. Can be operated under fluorescent lamp; Fast luminescence, fluorescence can make X-ray film sensitive for more than 12 hours, especially suitable for trace protein or nucleic acid detection; A higher antibody dilution ratio (1:2000 ~ 1:10,000) can be used to save antibodies.

Usage :

1. Perform conventional SDS-PAGE, transmembrane, and Western Blot procedures. Attention should be paid to using HRP to label lgG or monoclonal antibody - streptavidin - biotin -HRP clip method.

2. Prepare the luminescent working liquid freshly while washing the film for the last time by Western Blot: take solution A and B of equal volume respectively and mix them in a clean container. It is recommended to use the working liquid immediately, and it can still be used after several hours at room temperature, but the sensitivity is slightly reduced.

3. Remove the membrane with tweezers and place it on filter paper to drain the liquid but do not let the membrane dry completely. The film is completely immersed in the luminescent working liquid (0.125mL luminescent working liquid /cm2 film) and fully contacted with the luminescent working liquid. Incubate at room temperature for 3 minutes and prepare for immediate tablet exposure. Prolonged incubation does not increase sensitivity and sometimes results in abnormal exposure bands. The nature of the luminescence process is enzymatic reaction, and using too little luminescent working fluid will not be conducive to the reaction, which will also lead to uneven exposure of bands on the film and significantly reduce sensitivity. In order to achieve the purpose of saving, the film can be cut small but do not reduce the amount of luminous liquid.

4. Pick up the film with tweezers and place it on the filter paper to drain the luminescent working liquid. But do not wash away the luminescent liquid.

5. Open the X-ray film cassette and lay a piece of plastic wrap larger than the film on the inside surface of the cassette. Put the Western Blot film on the plastic wrap, fold the plastic wrap to completely cover the Western Blot film, remove bubbles and wrinkles, and cut off the excess plastic wrap at the edge. Remove excess luminescent working fluid with filter paper. Use tape to secure the plastic wrap covering the Western Blot film in the cassette, with the protein band facing upward.

6. Press the X-ray film in the darkroom, exposing it for different times such as seconds to minutes. Development rinse.

Note:

1. Steps 1 ~ 5 can be operated under fluorescent lamp; However, the sensitivity of the luminescent liquid exposed to strong light for too long may be slightly reduced, which can be avoided by moving to the darkroom. Wearing gloves can avoid leaving fingerprints on the membrane.

2. Long exposure time or excessive protein will deepen the background and make the band strength change lose linear relationship. Under exposure, the bands are blurred.

3. The bands on the film glow after the luminescent working liquid is incubated for about 3 minutes. Strong band luminescence is visible to the naked eye in the darkroom, and low abundance protein bands are weak and even invisible to the naked eye but can expose X-ray film. Band luminescence time cannot be judged simply by naked eye observation. The fluorescence, which is invisible to the naked eye, actually lasts for several hours and sensitizes the X-ray film, so the weak band can be exposed for 1-10 hours. If the banding is not good after exposure, the film can be washed with a wash film buffer, the secondary antibody is re-incubated, and then the ECL is re-illuminated and exposed.

4. Because the supersensitive luminescent liquid is extremely sensitive, it is strongly recommended that the starting concentration of most imported antibodies is 1:1000 ~ 1:4000 for the primary antibody and 1:2000 ~ 1:5000 for the second antibody. Too high an antibody concentration will cause a high background or no band, resulting in failure.

5. Some plastic wrap wrap imprinted film may quench fluorescence, should choose high-quality plastic wrap.

6. The position and size of the strips on the film can be precisely determined using visible predyed protein markers and fluorescin-autoradiography exposure labels.

7. NaN3 can inhibit HRP activity, and NaN3 should be avoided when recovering the second antibody, if necessary, no more than 0.01% should be used.

Note：Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.

Remark：These protocols are for reference only. Solarbio does not independently validate these methods.

Note：

1. The products are all for scientific research use only. Do not use it for medical, clinical diagnosis or treatment, food and cosmetics, etc. Do not store them in ordinary residential areas.

2. For your safety and health, please wear laboratory clothes, disposable gloves and masks.

3. The experimental results may be affected by many factors, after-sale service is limited to the product itself and does not involve other compensation.