Protein Protocols Protein Protocols

Chemifluorescence 421
56
Detection of Proteins on
Western Blots Using Chemifluorescence
Catherine Copse and Susan J. Fowler
1. Introduction
Immunodetection of specific proteins that have been immobilized on membrane supports
by Western blotting is a widely used protein analysis technique. Traditionally,
radioactively labeled antibodies or ligands (usually 125I) were used to probe for specific
antigens (1–3). Autoradiographic detection on X-ray film, for example, reveals
the location of the antigen with reproducibly high sensitivity and the results are easily
quantifiable. However, the need for lengthy exposure times and concerns related to the
handling and disposal of radioactive materials have led to the development of nonradioactive
techniques for Western blot analysis, for example, using colorimetric and chemiluminescent
detection.
In this case, probes can be labeled with reporter enzymes and when the appropriate
substrates are added, the position and quantity of antigen are indicated by the presence
and intensity of colored or luminescent products precipitated on the membrane at the
site of the immobilized enzyme. The two systems that are commonly used to achieve
this conversion are oxidation by horseradish peroxidase (HRP), or cleavage of a phosphate
group by alkaline phosphatase (AP). These enzymes can be conjugated directly
to an appropriate primary antibody or to a secondary antibody or ligand such as
streptavidin that binds to biotinylated primary antibodies.
An alternative detection strategy involves the use of antibodies or ligands linked
directly to fluorescent dyes, for example, fluorescein isothiocyanate (FITC) (1) or cyanine
dyes (4,5). The absorption of light energy by a fluorochrome causes excitation of
electrons to a higher energy state. These excited electrons spontaneously decay back
toward the ground state (lower energy level) within a few nanoseconds of excitation,
and as the electrons decay, the fluorochrome emits light of a characteristic spectrum.
Because the emitted light is always of longer wavelength (lower energy) than the excitation
light, optical filters can be used to separate excitation light from emitted light.
This allows the capture of the emitted light using imaging instruments that have the
appropriate excitation wavelength and emission filters for the particular fluorophore.
A more recent advance has been the development of chemical fluorescence detection
systems. Chemifluorescence relies on the enzymatic conversion of a nonfluorescent
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
421