Protein Protocols Protein Protocols

790 Gravel
dimethylformamide. These two solutions are stable when stored in closed containers
at room temperature.
b. AP buffer, composed of 100 mM NaCl, 5 mM MgCl 2, and 100 mM Tris-HCl, pH 9.5 is
prepared just before the colorimetric detection.
7. For the chemiluminescent detection of HRP activity, X-ray films (X-OMAT S, 18 × 24 cm)
and a cassette (X-OMATIC cassette, regular screens) are available from Kodak.
8. For the development of X-ray film, an automatic developer machine is used (Kodak RP
X-OMAT Processor, Model M6B). Manual development is also possible using the developer
for autoradiography films (ref. no. P-7042, Sigma) and the fixer (ref. no. P-7167, Sigma).
3. Method
The use of gloves is strongly recommended to prevent blot contamination.
1. After electrophoresis and protein blotting procedures, the membrane is first washed with
distilled water (3 × 5 min) and then treated for 1 h at room temperature and under gentle
agitation with 100 mL of blocking solution (PBS containing 0.5% [w/v] Tween 20) (see
Note 3).
2. The blot is then incubated for 2 h in biotinylated lectin, at a concentration of 1 µg/mL in
the blocking solution, under agitation, in a glass dish and at room temperature. Twentyfive
milliliters of lectin solution is used for the incubation of a membrane of 16.5 cm × 21 cm
(see Note 4).
3. A washing step is then performed for 1 h with six changes of 200 mL of PBS–Tween 20.
4. Extravidin-HRP or Extravidin-AP diluted 12000 in the blocking solution is added for 1 h
at room temperature under agitation. The membrane is then washed for 1 h with six
changes of PBS–Tween 20.
5. The colorimetric reaction with AP is carried out under gentle agitation by incubating the
blot in the following solution: 156 µL BCIP stock solution, 312 µL of NBT stock solution
in 50 mL of AP buffer. The colorimetric reaction is normally completed within 10–20 min.
The blotted proteins are colored in blue.
6. The chemiluminescence detection of peroxidase activity is performed according to the
manufacturer’s instructions (Amersham). The enhanced chemiluminescent assay involves
reacting peroxidase with a mixture of luminol, peroxide, and an enhancer such as phenol.
(See also Chapter 57.) Five milliliters of detection solution 1 are mixed with 5 mL of
detection solution 2 (supplied with the ECL Kit). The washed blot is placed in a glass
plate and the 10 mL of chemiluminescent reagents are added directly to the blot with a
10-mL pipet, to cover all the surface carrying the proteins. The blot is incubated for 1 min
at room temperature without agitation.
7. The excess chemiluminescent solution is drained off by holding the blot vertically. The
blot is then wrapped in plastic sheet (Saran Wrap), without introducing air bubbles and
exposed (protein side up) to X-ray film in a dark room, using red safelights. The exposure
time of the film depends on the amount of target proteins on the blot (see Note 5).
8. The development of the X-ray films can be done with an automatic developer or manually
with the following protocol:
a. The developer and fixer solutions are prepared according to the manufacturer’s
instructions (14 dilution with distilled water).
b. In a dark room, the film is attached to the film hanger and immersed in the developer
solution until the bands (or spots) appear (the maximum incubation time is 4 min). The
film should not be agitated during development.
c. The hanger is immersed into water and the film is rinsed for 30 s to 1 min.