Ultrasense? ECL Chemiluminescent Substrate Kit User Manual

Product

Name

specification

Reagent A

Luminescent Agent / Enhancer Solution

25ml

Reagent B

Peroxide buffer

25ml

composition:

Product introduction: ECL chemiluminescent substrate is a highly sensitive detection reagent used in conjunction with horseradish peroxidase (HRP) in immunoblotting experiments. This product undergoes a chemiluminescence reaction under the catalysis of HRP. It is used to detect proteins and DNA and other biological macromolecules fixed on the PVCF membrane. It can detect antigens at the level of 10-15g; the emitted light intensity is strong and durable, and can be used for photography Technology (X-ray film exposure), gel imager and other methods to display and test.

Storage conditions: -4 ℃, protected from light, the shelf life is two years.

Steps:

1. After incubation according to conventional cell membrane breaking, protein extraction, electrophoresis, membrane transfer, HRP-labeled antibody or nucleic acid probe, wash the blotting membrane thoroughly.

2. According to the required amount, freshly prepare the luminous working solution: take an equal volume of solution A and solution B, mix well and use immediately.

3. Remove the blotting membrane from the washing solution and drain the washing solution on the filter paper. Immerse the film in the luminous working fluid and make full contact with it. Incubate at room temperature for 2-5 minutes.

4. Remove the imprinted membrane from the luminescent solution and drain off excess luminescent working solution with filter paper.

5. Place a plastic wrap on the inside of the X-ray film cassette with an area larger than the blotting film. Place the hybrid membrane close to the plastic wrap to remove air bubbles and wrinkles.

6. Press the imprinted film on the X-ray film in a dark room and expose for different times, such as seconds to tens of minutes. Developer rinse.

Precautions:

1. In order to obtain the best experimental results, please be sure to optimize the experimental conditions, including the amount of test samples, the dilution of primary antibodies, secondary antibodies, and the choice of hybridization membrane and blocking reagents; it is recommended that the working concentration of primary antibodies be 50ng / ml-1μg / ml, the secondary antibody is 5ng / ml-50ng g / ml.

2. Because milk contains endogenous biotin, when using avidin / biotin system, please avoid using milk powder in the blocking solution;

3. Add high-quality Tween-20 (final concentration of 0.05%) to the blocking buffer and all antibody dilutions to reduce

Less non-specific binding;

4. Since sodium azide is an HRP inhibitor, avoid using sodium azide as a preservative in the buffer;

5. Make sure that the blotting membrane is always in a wet state during the entire immunoblotting experiment process to avoid drying;

6. In order to obtain the best experimental results, please use a shaker to assist in the antibody incubation and membrane washing steps;

7. Under dark conditions, the prepared chemiluminescent working solution can be stable for 8 hours at room temperature. Sunlight or other strong light will affect the effect of the working fluid. For best results, prepare working fluid in a brown bottle. Short-term exposure of normal laboratory lights does not affect the use of working fluids.

8. Excessive protein or long-term exposure will deepen the background and change the band strength to lose the linear relationship. Underexposure blurs the banding.

9. The band on the membrane glows after incubating for about 3 minutes with the luminous working solution. High-abundance protein bands are more luminous and visible to the naked eye in the darkroom. Low-abundance protein bands are weakly luminous and invisible to the naked eye, but can expose X-ray film. Therefore, the light-emitting time cannot be judged with the naked eye observation result. Sometimes the weak band needs to be exposed for 1-10 hours. If the band is not good after exposure, wash the membrane with washing buffer, re-incubate the secondary antibody, and re-emit luminescence and exposure.

10. Some commercially-available cling film will cause the fluorescence to be quenched when the imprinted film is wrapped. High-quality cling film should be selected.

Possible problems and solutions:

problem

possible reason

Solution

Film reversal (white stripe, black background)

Excessive HRP in the system

Dilute the HRP marker at least 10 times

Brown or yellow bands appear on the membrane

Strong light is seen in the dark room

Illuminated signal duration is too short

Weak or no signal

Excessive HRP in the luminescence reaction system consumes the substrate too fast, causing the signal to decrease rapidly

Dilute the HRP marker at least 10 times

Insufficient amount of antigen / antibody

Increase antigen / antibody usage

Low protein transfer rate

Optimized transfer system

High background

Excessive HRP in the system

Dilute the HRP marker at least 10 times

Insufficient closure

Optimize closed procedures

Improper selection of blocking reagent

Choose another blocking reagent

Insufficient flushing

Increase flushing time, times

Overexposed

Reduce exposure time

Antigen / antibody concentration is too high

Reduce antigen / antibody concentration

Protein band

Protein transfer failure

Optimize the transfer process

Membrane unbalance

Dispose of the membrane according to the instructions

There are air bubbles between the X-ray film and the film

Remove all bubbles before exposure

Non-specific bands appear (signal sustain time is short, high background)

Too much HRP in the system

Dilute HRP marker

Non-specific bands appear (signal maintenance time is normal, background is clean)

Too much primary antibody

Further dilution of the primary antibody

SDS leads to non-specific binding

Avoid using SDS during the experiment