ECL Western Blot Detection 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
Horseradish peroxidase (HRP)
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
kit price is lower than the cost of any ECL reagent from other
sensitivity; You could have the good results on the 5-40 ug/well
protein loading, exposure at 10-30 seconds see the result
any molecular Size, see below Fig.
Fig-1:Protein Loaded (ug /well)
Loaded (ug /well)
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
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
For 1 year
Western membrane in the western wash container with wash
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
*doing the process under the nature light. Or in dark Room
under the red safely light.
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.
off excess ECL mixture and wrap the membrane in plastic wrap.
Gently remove air bubbles.
the membrane, protein side up, in the film cassette, and bring
the cassette to dark room soon.
off the lights and use red safety light. Place a sheet of film
on the membrane, close the cassette and expose for 30-60
the second film for a suitable time according to the signal
intensity on the first film.
the signal was too low, you could keep the exposure at RT for
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.
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.