Protein Protocols Protein Protocols

Chemifluorescence 423
1. Like chemiluminescence, chemifluorescence is based on the affinity binding of an
enzyme-conjugated probe that acts to convert a substrate to product. Each enzyme conjugate
molecule can generate many fluorochromes. Under appropriate conditions, enzymes
can continue to convert substrate to product as long as the substrate is available. At the
lower limits of detection, it can be beneficial to allow the signal to develop for longer,
maximizing the signal from low abundance targets.
2. The signal does not decay in the same rapid time frame as chemiluminescence. Light
emission from a fluorochrome occurs only during exposure to excitation light, but a single
fluorochrome can be excited repeatedly and will emit light each time. As a consequence,
blots can be scanned many times.
3. If an initial image is too weak or the pixels are saturated, the sensitivity of detection can be
adjusted by changing the (PMT) voltage setting if acquired using a scanner, or by altering
the length of exposure if using a CCD camera.
4. The use of film and associated volatile and unpleasant development and fixing solutions
are avoided, together with the need for a dark room.
5. Scanning samples labeled with dyes excitable by the laser of an appropriate wavelength in a
fluorescence scanner or CCD camera enables images to be captured directly in digital format,
allowing for easier and more accurate quantification using image analysis software.
2. Materials
2.1. Solutions
1. Detection substrate:
1–5 µM DDAO-phosphate (Molecular Probes) in 10 mM Tris-HCl, 1 mM MgCl 2, pH 9.5.
2. Detection kits:
ECF Western blotting kits (Amersham Pharmacia Biotech).
Pro-Q TM Western Blot Stain Kit (Molecular Probes) DDAO-phosphate based (see Note 18).
FluoroBlot Peroxidase Substrate (Pierce Chemical).
ECL Plus (Amersham Pharmacia Biotech).
3. Phosphate-buffered saline (PBS), pH 7.5: 11.5 g of disodium hydrogen orthophosphate
anhydrous (80 mM), 2.96 g of sodium dihydrogen orthophosphate (20 mM), 5.84 g sodium
chloride, dilute to 1000 mL with distilled water and check pH.
4. Tris-buffered saline (TBS), pH 7.6: 20 mL of 1 M Tris-HCl, pH 7.6 (20 mM), 8 g of
sodium chloride (137 mM), dilute to 1000 mL with distilled water and check pH.
5. 0.1% PBS– and TBS–Tween TM 20 (PBST and TBST): Dilute the appropriate volume of
Tween 20 in PBS or TBS.
6. Blocking agent: PBST or TBST and blocking agent. Weigh out the appropriate amount of
blocking agent and dissolve in PBST or TBST as appropriate. The blocking agent can be
BSA, nonfat dried milk (5%), casein, fish gelatin, at between 1% and 5%, or various
cocktails thereof.
7. Stripping buffer: 100 mM 2-mercaptoethanol, 2% (w/v) sodium dodecyl sulfate, 62.4 mM
Tris-HCl, pH 6.7 (8).
Prepare all solutions using high-quality reagents and deionized water (18.2 MΩ
resistivity).
2.2. Equipment
1. Membrane: Polyvinylidene fluoride (PVDF), for example, Immobilon-P TM (Millipore),
Hybond TM -P (Amersham Pharmacia Biotech), or nitrocellulose, for example, Hybond
ECL (Amersham Pharmacia Biotech).