Protein Protocols Protein Protocols

218 Magi et al.
7. Fill the cell with transfer buffer and place a stir bar inside the transfer cell, so that the
buffer is stirred during electrotransfer (see Note 7).
8. Place the gel holder in the transfer cell with the sandwich oriented as follows: ANODE /
fiber pad, filter paper, nitrocellulose, gel, filter paper, fiber pad/ CATHODE.
9. Carry out blotting at a constant current until it is reached a total of 1.5–2.0 A° (see Note 8),
refrigerating the buffer to 4°C (see Note 9).
10. After electrotransfer, disassemble the blotting apparatus and remove the nitrocellulose
membrane. To mark the orientation of the membrane, cut away the lower right corner,
corresponding to low M r, high pH.
The membrane can be processed immediately for immunoblotting or can be airdried
and stored at –20°C, within parafilm sheets for extended periods (28).
3.2. Staining of Total Protein Pattern on Membrane
1. Before the immunodetection, stain the nitrocellulose membrane in 0.2% (w/v) Ponceau S
in 3% (w/v) trichloroacetic acid for 3 min (29) (see Note 10).
2. Destain with several changes of distilled water to diminish background color. Because the
red spots will disappear in the blocking step, circle with a waterproof pen some spots
which will be used as landmarks to match total protein pattern on nitrocellulose vs immunoreactive
pattern and vs silver stained polyacrylamide gel pattern (see Note 10).
3.3. Immunodetection
3.3.1. Incubation with Antibodies
All steps are carried out at room temperature and with gentle agitation on a rocking
agitator.
1. Block nonspecific binding sites in the membrane with three washing steps, each 10 min in
duration, in blocking solution (see Notes 11 and 12).
2. Incubate overnight in the primary antibody solution at the suitable dilution (see Note 13)
in blocking solution.
3. Wash 3 × 10 minutes in blocking solution.
4. Incubate for 2 h in the secondary antibody solution (see Note 14).
5. Wash 3 × 10 min in blocking solution.
6. Wash 30 min in washing solution.
7. Wash 2 × 30 min in 0.05 M Tris-HCl, pH 6.8.
After this step one can go forward with ECL detection (see Note 15). Alternatively,
one can choose detection with the chromogenic substrate (see Note 16).
3.3.2. Enhanced Chemiluminescent Detection
To detect the immunoreactive spot(s) with Amersham ECL kit, it is necessary to
work in a darkroom and to wear gloves to prevent hand contact with film.
1. Mix equal volumes of detection reagent 1 and detection reagent 2 from the Amersham
ECL kit and immerse the membrane in this solution for 1 min, ensuring that all the surface
of the membrane is covered with solution (see Note 17).
2. Place the membrane on a glass and cover it with a layer of Saran Wrap.
3. Cut away a corner from a piece of autoradiography film to define its orientation (see
Subheading 3.1., step 10). Superimpose the autoradiography film on the nitrocellulose
membrane beginning from the upper left corners. Nitrocellulose membrane and X-ray