Protein Protocols Protein Protocols

Electroelution of Proteins 301
3. Method
1. After electrophoresis, stain the gel for 15 min with Coomassie Blue. To prevent irreversible
fixation of the protein, destaining is observed on a light box. As soon as the protein of
interest becomes visible, the band is cut out with a razor blade. The gel piece is washed
once with 10-mL of 1 M Tris-HCl, pH 8.0 for 5 min, followed by three 10-mL washes
with water for 5 min each. Cut the gel piece with a razor blade into small cubes. Equilibrate
them in 10-mL of electroelution buffer for 10 min with occasional shaking. In the
meantime, assemble the electroelution apparatus.
2. The electroelution device is a block of polycarbonate (160 × 30 × 30 mm) that has an open
channel along its axis (Fig. 1). An elution chamber that holds the polyacrylamide pieces is
formed with trap inserts between points C and F of the body. During the elution process,
the protein is trapped into a chamber formed between points F and G (Fig. 1). The device
works with two types of membranes having different ion permeabilities: the BT1 membrane
retains all charged macromolecules larger than 5 kDa, whereas buffer ions can freely
permeate under the influence of an electric field. The macroporous BT2 membrane acts as
a barrier that prevents particulate matter from entering the trap. It also keeps the buffer
from flowing into the trap when the electric field is switched off, preventing dilution of
the protein in the trap.
Slide BT1 membranes between the clamping plates and the trap inserts at positions A and
G of the BIOTRAP apparatus (Fig. 1). Since the BT1 membrane is an asymmetric membrane
with two different surfaces, make sure that they are mounted in the proper orientation.
The BT1 membrane is delivered moist and should not dry out. Buffer should be
added within 5 min after insertion of the membranes. Insert a BT2 membrane at points
C and F. If necessary, smaller elution chambers can be formed by inserting the BT2 membrane
at positions D or E. Tighten the pressure screws to hold the membranes in place.
Transfer the gel pieces with a spatula into the elution chamber formed between the two
BT2 membranes inserted at positions C and F. Carefully overlay the gel pieces with
electroelution buffer until the level of the liquid is approx 5 mm above the gel pieces.
After some minutes, the trap is filled with buffer by seeping through membrane BT2.
Make sure enough liquid is in the elution chamber so that the gel pieces remain completely
immersed. Place the electroelution device into a horizontal electrophoresis chamber
with the + mark directed toward the anode of the electrophoresis chamber. The
dimensions of the horizontal electrophoresis tank are as follows: 30 cm length, 20 cm
width, and 7 cm depth. The T-shaped table for agarose gels is 3 cm from the bottom. Add
enough electroelution buffer to the electrophoresis chamber to fill half of the BIOTRAP
(approx 3 L). Electroelute the protein for 18 h at 100 V (the current will be in the range of
70–90 mA). The volume into which the eluted protein is recovered depends on the buffer
level inside the BIOTRAP and ranges from 200–800 µL.
3. Replace the electroelution buffer with 3 L of electrodialysis buffer and electrodialyze the
sample for 6 h at 40 V against 15 mM NH4HCO3, 0.025% SDS.
4. Remove the eluted protein from the trap. Be careful not to perforate the BT1 membrane
with the pipet tip! Rinse the trap twice with 100 µL of fresh electrodialysis buffer. Combine
the dialysate and the washes. The solution is dried in a Speed Vac concentrator and
stored at –20°C.
5. For desalting of the electroeluted protein, equilibrate the PHA column with solvent A
(65% n-propanol, 50 mM formic acid). Electroeluates containing > 5 µg of protein are
desalted on 4.6 mm internal diameter columns, which are operated at a flow rate of
0.5 mL/min. Less than 5 µg protein are chromatographed on 2.1 mm internal diameter
columns at 75 µL/min. The effluent is monitored at 280 nm.