Protocol for

Comparison of Three Commonly Used Chemiluminescent Substrates

Link to Full Protocol: http://www.carestreamhealth.com/common-chemiluminescent-AP00 ...

Abstract or Description:

The most widely used detection method for Western blotting is enhanced chemiluminescence where horseradish peroxidase (HRP), conjugated to the secondary antibody, catalyzes the oxidation of luminol in the presence of hydrogen peroxide. This reaction emits light at 428 nm, which can be captured on film or using a digital imager with a CCD camera.

There are many variations in the chemical formulation of chemiluminescent substrates that optimize different components of the enzymatic reaction. Thus, substrates in general demonstrate a wide range in detection sensitivity, duration of signal output, compatibility with different film or digital imaging systems, and price. The most sensitive reagents are more expensive, and require optimization to reduce non-specific background, but provide femtogram-level sensitivity for low abundance protein targets. The less expensive substrates are not as sensitive, detecting down to several picograms.

The duration of signal can also be an important factor in choosing the correct chemiluminescent substrate because a longer duration signal allows for the opportunity to capture multiple exposures on a digital imager, to acquire the best image before the chemiluminescent reaction has diminished.

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Posted By: Carestream Health on 1/28/2011 11:02:56 AM

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