Enhanced chemiluminescence (ECL) is a method which provides highly precise detection of proteins from Western blots. For a description of Western Blotting see our protocol.
In Western blotting we immobilized our proteins on a membrane and labeled them by complexing them with primary antibodies followed by horseradish peroxidase (HRP) conjugated secondary antibodies. To develop our Western blots we use an enhanced version of the chemiluminescence reaction.
In the chemiluminescence reaction horseradish peroxidase catalyzes the oxidation of luminol into a reagent which emits light when it decays. Since the oxidation of luminol is catalyzed by horseradish peroxidase, and the HRP is complexed with the protein of interest on the membrane, the amount and location of light that HRP catalyzes the emission of, is directly correlated with the location and amount of protein on the membrane.
In many luminescenct assays, the light emitted is of low intensity and short duration. We use an enhanced version of the chemiluminescence reaction. This involves performing the reaction with luminol in the presence of modified phenols. The phenols greatly enhance light emission,
altering the kinetics of the reaction in such a way that the light emission is much prolonged and very intense.
With enhanced chemiluminescence the phenol enhanced luminol reagent increases the light emission more than 1000-fold*. This allows detection of protein at a level of 1-10 pg. When the enhanced luminol reagent degrades, light emission occurs at a wavelength of 428 nm. This light can be quickly captured in hardcopy on Bioflex Scientific Imaging film, or on the KODAK Image Station.
Materials for this procedure are listed at the end of this page. Also refer to the PerkinElmer® Western Lightning® Chemiluminescence Reagent Plus manual.
* Thorpe, G. H. G., Kricka, L. J., Mosely, S. B. and Whitehead, T. P. Phenols as enhancers of the chemiluminescent horseradish peroxidase-luminol-hydrogen peroxide reaction: Application in luminescence-monitored enzyme immunoassays. Clin. Chem. 31:1335-1341(1985).
Follow the protocol for Western blotting. At the end you will have your blot in a weigh dish at your lab bench.
In a beaker prepare the Western Lightning® Chemiluminescence Reagent Plus system
by mixing equal volumes of Enhanced Luminol Reagent (brown bottle) and the Oxidizing Reagent (white bottle).
Immediately pour the chemiluminescence reagent into the weigh dish with the membrane and incubate for 1 minute at room temperature. Use at least 0.125 mL per cm2 membrane and incubate with shaking.
Remove excess chemiluminescence reagent by covering membrane with extra blotting paper for 5-10 sec.
Developing Blots with Scientific Imaging Film
Take the blot to the developing room and place the membrane between the covers of a propylene sheet protector with the black interface removed (plastic wrap works well also). Gently smooth out any air pockets.
Switch off the lights and place the Kodak Scientific Imaging film on top of the membrane.
Expose the film for 30 seconds, then develop.
Repeat the exposure, varying the time as needed for optimal detection.
Analyze developed film using ImageJ software.
Visualization of Western Blots on the Kodak Image Station
Take blot to the the Kodak Image Station system and turn it on.
Set the camera for optimal western blot image capture conditions (Fstop: 1.2, zoom: 20, no filter, focus: 1.8).
Take ten captures of the images at optimal exposure times for the specific antibody (PARP: 1.5 minutes, BclXL: 1.5 minutes, Bcl2: 35 seconds and actin: ~5 seconds).
Perform analysis of the band intensities using the associated Image Analysis Software. Adjust captured images for optimal contrast for viewing, and manually acquire unadjusted band intensities by selecting lanes, bands and background using the Kodak Image Station software. Record net band intensities using the “net intensity” selection in the Image Analysis software, which automatically adjusts for selected background readings.
Enter the results into an MS Excel (Microsoft) spreadsheet for graphical and statistical analysis.
Chemiluminescence Reagent Bottle 1
Chemiluminescence Reagent Bottle 2
Propylene sheet protector with the black interface removed / plastic wrap