Biomedical Engineering Reference
In-Depth Information
2.5 Electroelution fromGels
2.5.1 Preparative Electroelution of Proteins
from Polyacrylamide Gels
This protocol is designed for proteins separated by SDS-containing
PAGE systems. When proteins shall be eluted from gels of other
systems, the respective electrode buffer has to be used instead of
Soln. A and the polarity should be taken into account.
The content of glycerol or sucrose in Soln. A complicates the
removal of SDS; therefore, the sample has to be dialyzed after
electroelution. To avoid difficulties use Soln. A without glycerol or
sucrose.
A 5mMTr i s , 0 . 2 Mglycine, 0.1% SDS (w/v), pH 8.3 [the solution
Solutions/Reagents
may contain glycerol or sucrose up to 30% (w/v)]
B 0mM ammonium hydrogencarbonate buffer, pH 7.5; con-
tains maximal 0.05% SDS if necessary
C 80% TCA (w/v) in ddH
2
O
D1mM HCl in acetone
Acetone
The gel slices containing the protein of interest are minced in the
presence of Soln. A (buffer volume nearly the same of gel volume),
filled into a vessel of appropriate size and sonicated for 30 - 60 min.
Use an electrophoresis tube with an inserted sintered glass or
polymer disk about 1 mm above one end and fill it at this side
with buffer. Cover with a dialysis membrane with appropriate M
r
cut-off, and fix with a rubber ring
19
.
Fill the gel particles together with the surrounding buffer into
the tube. The tube is inserted into the vertical electrophoresis ap-
paratus (dialysis membrane down to anode), electrode buffer is
poured, and electroelution is started with 10 mA per tube for 3 -
8 h, depending on the molar mass of the protein.
The eluted protein is taken off by a syringe from the space
between sintered glass and dialysis membrane.
If the eluted protein is used for amino acid analysis, the sam-
ple has to dialyze three times 1 h each against 100-fold of Soln. B.
After lyophilization, the protein is analyzed. SDS, which is not
dialyzable under the given conditions, does not influence the de-
termination.
If the protein concentration of the eluate is too low for fur-
ther investigation, concentrate the sample using a centrifuge mem-
brane concentrator (Centricon) with molar mass cut-off signifi-
cantly lower than the M
r
of the analyzed protein or by precipitation
with TCA or acetone.
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Electro-eluter are commercially available. Their only limit is fixed vol-
umes for gel and eluate.
