Protein Protocols Protein Protocols

430 Dickinson and Fowler
the advantage that the signal is read directly from the bound antibody but offers lower
sensitivity (5) than chemifluorescence or chemiluminescence. Chemifluoresence (6) is
analogous to chemiluminescence in utilizing an enzyme to cleave a substrate but generating
a fluorescent product rather than light in the process.
Light emitting chemiluminescence detection has become extremely popular over
the last 10 yr and many reagents are now available. Utilization of substrates for alkaline
phosphatase produces hard copy on film with good sensitivity, but can sometimes
suffer from background problems due to bacterial alkaline phosphatase contamination
of buffers. Chemiluminescent systems based on horseradish peroxidase (HRP) offer
rapid highly sensitive results with excellent signal-to-noise ratio and have become the
predominantly used detection method. There are two different substrate types available,
those based on the oxidation of luminol by HRP–hydrogen peroxide in the presence
of phenolic enhancers (7) (e.g., ECL TM ), and more recently, those based on the
oxidation of acridan substrates (8) (e.g., ECL Plus TM ). During the course of the ECL
Plus reaction, fluorescent intermediates are generated, allowing signal to be measured
using fluorescence scanners. As an example, ECL Plus detected using three imaging
techniques—film plus densitometry, charge coupled device (CCD) camera detection,
and fluorescence scanning—is described in this chapter.
Western blots detected with chemiluminescent substrates are typically quantified
using images generated by autoradiography film followed by digitization on a densitometer.
Although film itself has a dynamic range of 5 × 10 2 , in practice the linear range
over which proteins can be quantified has been estimated to be just over one order of
magnitude (9). Film-based detection suffers several drawbacks. At low levels of target,
film exhibits reciprocity failure, which means it requires a threshold level of signal
before an image is generated. Preflashing of the film to the detection threshold can
overcome this to some extent and improve linearity (10). At high target levels, saturation
effects mean film darkening is again not linear with respect to the amount of light
produced. For this reason, when using film, it is important that several exposures are
taken to ensure the signal from the blot falls within the linear range of the film.
An alternative approach is to use a CCD camera to capture the chemiluminescent
light output directly. CCD cameras work by converting the light emitted from the blot
into an electrical signal which is then subsequently converted to a digital signal to
obtain a quantifiable value (11). For low level light imaging, cooled CCD cameras are
necessary to reduce background noise to obtain improved signal to noise and allow
longer exposures. Typically Peltier cooling is used, reducing the camera operating temperature
to –20°C to –60°C below ambient. Some cameras are capable of providing
equal or better sensitivity than that obtained with film. In addition, CCD cameras can
offer excellent dynamic range (10 3 –10 5 ) allowing accurate quantification.
An equally suitable alternative imaging method for quantification with ECL Plus is
to detect the fluorescent signal. This can be achieved using a flat bed laser scanner or
CCD camera coupled with a suitable excitation light source. Laser based fluorescence
scanners also offer excellent dynamic range (10 5 ) and the convenience of direct digitization
of signal. When imaging fluorescent blots, to obtain maximum sensitivity, it is
important the excitation light of the imager is well matched with the excitation wavelength
of the fluorescent product and that appropriate emission filters are used.