West Femto ECL Substrate

Product Introduction:

West Femto ECL supersensitive luminescent solution is used to detect antibodies and associated antigens that directly or indirectly label HRP. Thanks to its unique luminescent substrate system, West Femto ECL supersensitive Luminescent Solution is the most sensitive commercially available fluorescent ECL detection reagent. With extremely high sensitivity and high signal-to-noise ratio, it can detect 10 ~ 100 fg antigens. Luminous operation under fluorescent lamp. Rapid luminescence, fluorescence can make the 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.

Use: For Western Blot of HRP labeled antibodies and nucleic acid hybridization of HRP labeled probes.

Usage:

(1) Perform routine 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) The luminescent working liquid was prepared fresh while washing the film for the last time by Western Blot: equal volumes of solution A and B were respectively taken and mixed 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 film with tweezers, put it on the filter paper to drain the liquid but do not let the film 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 put 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) The X-ray film is pressed in the darkroom, and the exposure time is different, such as several seconds to several 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 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 ~ 1:400 for the primary antibody and 1:2000 ~ 1:500 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 location 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 the use of NaN3 should be avoided in the recovery of the second antibody, if necessary, do not exceed 0.01%.

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.