Far Western Blotting Protocol

Far Western Blot

Enhanced chemiluminescence (ECL) is a method which provides highly precise detection of proteins from Western blots.

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. 

ECL Western Blot Protocol - Critical Steps in ECL-Western Analysis

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.

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.

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.

Far western blotting