Protein Protocols Protein Protocols

338 Delcuve and Davie
of Tris base in 800 mL of water and adjusted to pH 6.8 with hydrochloric acid. The buffer
is made up to 1000 mL.
3. Transfer membrane: Nitrocellulose membranes (0.45-µm pore size, Schleicher & Schuell
[Keene, NH], BA85) are used.
4. Transfer buffer: 187 mL of Caps (3-[cyclohexylamino]-1-propanesulfonic acid) stock (16X)
solution (final concentration, 25 mM), 600 mL of methanol (20%), and water to 3 L. Caps
stock solution (400 mM): 88.5 g of Caps dissolved in 800 mL of water and adjusted to
pH 10.0 with 10 M NaOH. The buffer is made up to 1000 mL.
5. Electroblotting equipment: Proteins are transferred with the Bio-Rad (Hercules, CA)
Trans-Blot transfer cell containing a super cooling coil (Bio-Rad). Cooling is achieved
with a Lauda circulating bath.
6. Protein stain: Proteins in the AUT or SDS polyacrylamide gel are stained with Coomassie
blue (Serva Blue G). Proteins on the membrane are stained with Indian ink (Osmiroid
International Ltd.) (0.01% v/v in TBS-TW). TBS-TW: 20 mM Tris-HCl, pH 7.5, 500 mM
NaCl, and 0.03% (v/v) Tween-20.
3. Methods
3.1. Protein Blotting
1. Nitrocellulose membranes are cut to size of gel at least a day before transfer and stored in
water at 4°C.
2. The proteins are electrophoretically resolved on AUT- or SDS-polyacrylamide minislab
gels (6 mm long, 10 mm wide, 0.8–1.0 mm thick).
3. Following electrophoresis, the AUT polyacrylamide gel is gently shaken in 100 mL of
equilibration buffer 1 for 30 min at room temperature. This solution is poured off, and
another 100 mL of equilibration buffer 1 are added. The gel is again shaken for 30 min.
The solution is discarded and replaced with equilibration buffer 2. The gel is agitated in
this solution for 30 min.
4. Onto the gel holder, place the porous pad that is equilibrated with transfer buffer. Three
sheets of 3MM paper soaked in transfer buffer are placed on top of the porous pad. The
treated AUT or SDS slab gel is placed onto the 3MM paper sheets. Nitrocellulose membrane
is placed carefully on top of the gel, avoiding the trapping of air between the gel and
nitrocellulose membrane. One sheet of 3MM paper soaked in transfer buffer is placed on
top of the nitrocellulose membrane, followed by the placement of a porous pad that has
been equilibrated with transfer buffer.
5. The gel holder is put into the transblot tank with the polyacrylamide gel facing the cathode
and the nitrocellulose membrane facing the anode. The tank is filled with transfer buffer.
Protein transfer is carried out at 70 V for 2 h and/or at 30 V overnight with cooling at 4°C.
6. Following transfer, the nitrocellulose membrane is placed onto a sheet of 3MM paper and
allowed to dry for 30 min at room temperature. The gel is stained with Coomassie blue.
The air-dried nitrocellulose membrane is placed between two sheets of 3MM paper. This
is wrapped with aluminum foil and baked at 65°C for 30 min. The proteins transferred
onto the nitrocellulose membrane may be visualized by staining the nitrocellulose membrane
with India ink (see Subheading 3.2.2.).
3.2. Protein Staining
1. The nitrocellulose membrane is agitated in TBS-TW (0.7 mL per cm2 ) for 10 min at room
temperature in a sealed plastic box. The solution is discarded, and fresh TBS-TW is added.
These steps are repeated twice more for a total of four washes of the membrane in the
TBS-TW solution.