Protein Protocols Protein Protocols

Electroelution of Proteins 303
Fig. 2. Removal of SDS from an electroeluate. (A) 20 µg of a 45-kDa mitochondrial outer
membrane protein in 50 µL was injected onto a PHA column (4.6 × 200 mm) that had been
equilibrated in 70% n-propanol–50 mM formic acid. After the baseline had stabilized, the
gradient was initiated (marked with an arrow). Bound protein was eluted with a linear
10-min gradient from 70% n-propanol–50 mM formic acid to 50 mM formic acid at a flow rate of
0.5 mL/min. The protein eluted between 27 and 30 min. (B) Fractions of 500 µL were collected
and tested with Fuchsin red for the presence of SDS. (Modified with permission from ref. 7).
protein–SDS complexes with potassium (10), or precipitation of SDS by 4 M sodium
acetate (11). We found staining of complex protein patterns with these methods difficult,
as they tend to produce diffuse bands. Staining the gel for 15 min with Coomassie Blue
is sufficient to visualize also faint bands without fixing the protein irreversibly. To
minimize fixation of proteins, destaining is carried out on a light box, so that the band of
interest can be sliced out of the gel as soon as it becomes visible.
2. The electroelution apparatus routinely used in our laboratory was originally described by
Jacobs and Clad (12) and is commercially available from Schleicher and Schuell. We
found this type of apparatus very reliable for routine use. The volume of the elution chamber
can be adjusted depending on the volume of the gel pieces used. The volume can be
increased or decreased by varying the position of the BT2 membranes between positions
C and F (see Fig. 1). By forming the smallest possible elution chamber, one can process
Coomassie Blue-stained bands from a single one-dimensional analytical PAGE. With the
larger elution chamber, up to five preparative gels can be processed at a time. However,
other suitably constructed devices will give identical results.
3. So far we have eluted proteins between 20 and 100 kDa with protein amounts ranging
from 10 to 50 µg per band. After elution and dialysis, proteins are typically recovered in
volumes of between 300 and 800 µL. However, elution of