Protein Protocols Protein Protocols

388 Hong et al.
2. Materials
2.1. General
Fig. 1. Chemical structure of DB71.
1. Transfer apparatus.
2. Power supply (capacity 200 V, 2 A): Preferably capable of delivering constant current.
3. Nitrocellulose or PVDF membrane.
4. Filter papers (e.g., Whatmann no. 1).
5. Rocking shaker.
6. Plastic or glass container.
7. Transfer buffer: 25 mM Tris-HCl, pH 9.4, containing 20% methanol. Many different
buffers can be used in transferring proteins from gels, depending on the efficacy of
the blotting.
8. Dot or slot blot apparatus.
9. Phosphate buffered saline (PBS): Needed for dot or slot blotting. For 1 L of solution, use
0.2 g of KH 2PO 4, 1.15 g of Na 2HPO 4, 0.2 g of KCl, and 8.0 g of NaCl.
2.2. DB71 Staining
1. Stock dye solution (0.1% [w/v] DB71): Dissolve 0.1 g of DB71 (dye content, ~50%; from
Aldrich) in 100 mL of distilled water. The stock solution is stable for months at room
temperature.
2. Working dye solution (0.008% [w/v] DB71): Dilute 4 mL of stock dye solution to 50 mL
of washing solution (see ref. 3). The working solution is stable for weeks at room temperature.
3. Washing solution: For 1 L of solution, mix 500 mL of distilled water with 400 mL of
absolute ethanol and 100 mL of glacial acetic acid.
4. Destaining solution: For 1 L of solution, mix 350 mL of distilled water with 500 mL of
absolute ethanol and 150 mL of 1 M sodium bicarbonate.
3. Methods
3.1. Electrophoresis and Blotting
All procedures refer to product information of the apparatus used and general recipes.
1. Electrophorese proteins diluted to the appropriate concentrations according to Laemmli
(19) on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels.
2. Transfer proteins from gels onto membranes in a semi-dry transfer kit at a constant current
of 1 mA/cm 2 for 1–2 h, in 25 mM Tris-HCl –20% methanol, pH 9.4.
3. (Optionally) Make dot or slot blots with a Bio-Dot ® microfiltration apparatus (Bio-Rad
Lab., Richmond, CA).