ECL Western Blot Detection Kit 

Cat. No:  E-0010                                          Price: $129/kit 

ECL Western Blot Kit is a ready substrate to be widely used for chemiluminesecent detection on Western blot- immobilized protein conjugated with HRP directly.

Principle of ECL detection

In the presence of hydrogen peroxide(H2O2),), Horseradish peroxidase (HRP) catalyzes

The oxidation of cyclic diacylhydrazides, such as luminal. Following the oxidation, the luminal, as the intermediate reaction product, is in an excited state, which decays to the ground state by Emitting light. The strong enhancement of the light emission is produced by enhancers, such as Phenolic compounds. According to the principle, a new formulation,  the ECL Western Kit is designed to detect membrane immobilized specific antigen.

Highlights

1. The kit price is lower than the cost of any ECL reagent from other company.
2. High sensitivity; You could have the good results on the 5-40 ug/well protein loading, exposure at 10-30 seconds see the result below Fig.
3. And any molecular Size, see below Fig. too;

                     Fig-1:Protein Loaded (ug /well)              Fig.-2: Protein Loaded (ug /well)

                                  5     10      20     40                                       10        20         40

 Exposure                                                                       

10 seconds                 

20 seconds    

30 seconds  

The protein was from 293 cells, which were separated by electrophoresis and  transferred to nitrocellulose membranes. The membranes (Fig-1) were incubated with anti-Actin (42 Kda), Fig.-2 was incubated with 

Anti- Sirt1 (120 Kda), no blocked, at RT for 30 Minutes, then, were incubated with HRP-conjugated IgG at RT. Each membrane was detected with ECL Western Blot Detection Kit.

Content Table   

Quick Protocol:

1. Keep Western membrane in the western wash container with wash buffer.  

2. Mix an equal volume mixture of ECL Reagent 1 and 2 to give enough solution to cover the membrane (0.15-0.2 ml/cm²). Let the mixture equilibrate for 5-10 seconds. It is ready ECL mixture.

    *doing the process under the nature light. Or in dark Room under the red safely light.

3. Remove the wash buffer from the western wash container, and make sure the protein side is up, add the ready ECL mixture to the container immediately, shaking at RT for 30-60 seconds.

4. Drain off excess ECL mixture and wrap the membrane in plastic wrap. Gently remove air bubbles.

5. Place the membrane, protein side up, in the film cassette, and bring the cassette to dark room soon.

6. Switch off the lights and use red safety light. Place a sheet of film on the membrane, close the cassette and expose for 30-60 seconds.

7. Expose the second film for a suitable time according to the signal intensity on the first film.

8. If the signal was too low, you could keep the exposure at RT for 30 minutes.

9. If the signal intensity was too high, wait up to 30 minutes before re-exposing. If you know some antibody with strong signal, you could get the desired result by the ECL mixture was diluted with western wash buffer.

References:

1. Tsukagoshi K, et al.: Enhancing effect of phenylboronic acid compounds and their interactions with the diol groups of saccharides in a capillary electrophoresis-chemiluminescence detection system. Anal Sci., 2007 Feb; 23(2): 227-30.

2. Alpeeva IS, rt al.; Luminol-hydrogen peroxide chemiluminescence produced by sweet potato peroxidase. Luminescence, 2007 Mar-Apr; 22(2): 96-6.                                                                                                                      

                                    This product is distributed for laboratory research only.Caution: Not for diagnostic use.