Protein Protocols Protein Protocols

Immunoblotting of 2-DE Separated Proteins 217
2. Materials
2.1. Equipment
1. Blotting apparatus: Transfer cell, gel holder, magnetic stirrer, refrigerated thermostatic
circulator unit.
2. Power supply.
3. Rocking agitator.
4. Computing densitometer.
5. Workstation with a computer program for 2-D gel analysis.
2.2. Reagents
1. Distilled water.
2. Nitrocellulose membrane.
3. Transfer buffer: 25 mM Tris, 192 mM glycine, 20% (v/v) methanol. Do not adjust pH; it is
about 8.3.
4. Filter paper for blotting (Whatman 17 Chr).
5. Ponceau S solution: 0.2% (w/v) Ponceau S in 3% (w/v) trichloroacetic acid (TCA).
6. Phosphate-buffered saline (PBS): 0.15 M NaCl, 10 mM NaH2PO4, bring to pH 7.4 with
NaOH.
7. Blocking solution: 3% (w/v) nonfat dry milk in PBS, Triton X-100 0.1% (w/v).
8. Primary antibody solution (primary antibody, appropriately diluted in blocking solution).
9. Secondary antibody solution (secondary antibody, appropriately diluted in blocking solution).
10. Washing solution: Triton X-100 0.5% (w/v) in PBS.
11. 0.05 M Tris-HCl, pH 6.8.
12. Amersham ECL (enhanced chemiluminescence) kit, cat. no. RPN 2106.
13. Saran Wrap or other cling films.
14. X-ray films, 18 cm × 24 cm (Amersham Hyper film ECL, cat. no. RPN 3103).
15. Developer and fixer for X-ray film (Developer replenisher; fixer and replenisher, 3M, cat.
nos. XAF 3 and XAD 3) (3M Italia S.p.A., Segrate, Italy).
16. Stripping buffer: 100 mM 2-mercaptoethanol, 2% (w/v) SDS, 62.5 mM Tris-HCl pH 6.7.
3. Methods
3.1. Transfer
To avoid membrane contamination wear gloves during all the steps of the experiment.
1. Prepare the transfer buffer and cool it to 4°C before the end of the electrophoretic run (see
Notes 1–3).
2. Cut to the dimensions of the gel, two pieces of filter paper and one piece of nitrocellulose/
gel (see Note 4).
3. Following electrophoresis, wash the gel in distilled water and then equilibrate it in transfer
buffer. The ideal time for 1.5-mm gels is 10–15 min. (see Note 5).
4. Soak the nitrocellulose membrane for 15–20 min in transfer buffer. Also wet two “Scotch-
Brite” pads/gel and filter papers in transfer buffer.
5. Assemble the “sandwich” for transfer in this order: fiber pad, filter paper, nitrocellulose,
gel, filter paper, fiber pad. Remove all air bubbles between membrane and gel and between
paper and gel.
6. Put the blot sandwich in the gel holder and hold it firmly, to ensure a tight contact between
gel and membrane (see Note 6).