Protein Protocols Protein Protocols

422 Copse and Fowler
substrate to a fluorescent product, or the production of a measurable change in the
spectral properties of the substrate. As with chemiluminescence, this is usually
achieved by oxidation or dephosphorylation by HRP or AP, respectively. This technique
offers a signal amplification advantage over direct fluorescent labeling as each
enzyme molecule can convert multiple substrate molecules into fluorescent products.
The fluorescent product must also be immobilized at the site of enzyme activity on the
membrane to ensure correct imaging of the protein sample. This is achieved by a combination
of precipitation of the product and charge interaction with the membrane.
Fluorescence is detected by exciting the membrane with light of the appropriate
wavelength, from a broad wavelength source (ultraviolet [UV] or xenon arc lamps) or
using discrete wavelength excitation with a laser based fluorescence scanner. The emission
can be captured directly using a fluorescence scanner, equipped with a photomultiplier
tube (PMT), or, using a charge coupled device (CCD) camera. After the emitted
light is captured, the analog signal from a PMT or CCD is converted into a digital
signal. Alternatively, a Polaroid TM camera can be used to provide a hard copy of the
results. Chemifluorescence can be used in place of standard chemiluminescence protocols
but for optimal sensitivity it is important to use imaging equipment that closely
matches the excitation and emission wavelengths of the substrate.
Currently available chemifluorescent alkaline phosphatase substrates include
DDAO-phosphate (Molecular Probes) in which the phosphate group is cleaved to yield
a product with an excitation maximum of 646 nm and an emission maximum of
660 nm. The ECF TM (Enhanced Chemifluorescence) substrate available from
Amersham Pharmacia Biotech also has a phosphate group that can be cleaved by AP to
yield a product with fluorescence maxima of 435 nm for excitation and 555 nm for
emission. Kits containing the substrate and working buffer plus AP-conjugated
secondary antimouse and antirabbit antibodies are available. Figure 1 shows a typical
Western blot detected using ECF substrate. Alternatively a three-tier system is offered
in which the secondary antibody is conjugated to fluorescein. This allows direct
visualization of the fluorescein fluorochrome (maximum excitation 494 nm and
maximum emission 518 nm), but where greater sensitivity is required, tertiary
AP-conjugated anti-fluorescein antibodies can be used and the ECF substrate applied
to yield a chemifluorescent product.
Currently available peroxidase substrates yielding fluorescent products include
FluoroBlot TM Peroxidase Substrate (Pierce Chemical) and ECL Plus TM (Amersham
Pharmacia Biotech). The addition of the FluoroBlot working substrate to HRP, immobilized
on a membrane, results in the generation of a blue fluorescent product (excitation
maximum 325 nm, emission maximum 420 nm), which can be quantified using a
CCD or Polaroid camera and UV excitation. Finally, ECL Plus is a highly sensitive
chemiluminescent substrate system that utilizes a novel acridan-based chemistry (6) to
generate a light signal. However, it also produces a stable fluorescent intermediate that
can be detected with an excitation maximum of 430 nm and emission maximum of
503 nm on a membrane (7). It can be visualized using the Storm TM (Amersham
Pharmacia Biotech) fluorescence scanner with excitation at 450 nm.
1.1. Advantages of Chemifluorescent Detection
Chemifluorescence shares many of the advantages of chemiluminescence and has
additional features: