Bio-Rad Model 422 Electro-Eluter - Manual (Page 8)

5. Fill each tube with elution buffer and place the gel slice in the tube. To increase sample
recovery, several bands may be excised from the gel and minced, so long as the height of
the gel within the glass tube is approximately one centimeter or less. If the glass tube is
filled higher than 1 cm, elution times may increase.
6. Place the entire module into the buffer chamber. Fill the lower buffer chamber with ~600
ml of elution buffer. The level of the lower buffer must be above the top of the silicone
adaptors or bubbles may form on the bottom of the dialysis membrane. Fill the upper
buffer chamber with ~100 ml of elution buffer. To prevent electrical shorts the banana
plugs must be dry.
7. Place a stir bar in the bottom buffer tank. Vigorous stirring during the run will prevent bub-
bles from sticking to the bottom of the dialysis membrane.
8. Attach the lid with cables, orienting the red to red (anode) and black to black (cathode).
9. Elution is done at 8-10 mA/glass tube. See Sections 6 and 8 for specific conditions for elut-
ing protein and DNA. See Section 9 for elution buffer recipes.
Section 5
Protein Visualization in SDS-Polyacrylamide Gels
After the protein has been separated by gel electrophoresis, it is necessary to visualize
the band of interest. If the protein can be fixed, stained, and eluted see Section 6.1. Unstained
proteins can be isolated using one of the methods in Section 5.
5.1 Visualizing the Protein Using Coomassie Blue R-250
Using this method, a reference lane containing the same sample as the one being eluted
is run on the gel and stained to identify the band of interest. Prior to electrophoresis, the ref-
erence lane and the lane that will be eluted are treated exactly the same. The sample is prepared
as one large batch and equal amounts are loaded on the elution lane and the reference lane.
1. The reference lane should be run next to the elution lane. It should not be run on the end
lane because edge effects may distort the banding pattern. The gel is run at the optimal con-
ditions for the band of interest.
2. At the end of the run, cut a corner off the gel to maintain the correct orientation. Keep the
gel moist in a small amount of running buffer.
3. The reference lane is cut off, fixed, and stained with 10% acetic acid, 40% methanol,
0.1% Coomassie R-250 for 1 hour. It is destained using 10% acetic acid, 40% methanol
for 2 hours, or until the band of interest can be identified. It is not necessary to have a
completely clear background. The proteins will not diffuse in the 2-3 hours needed to do
the staining. Line up the stained gel lane with the unstained gel, making sure the corner
that was cut off is in the correct orientation. Cut out the appropriate area of the unstained
gel along with some extra gel above and below the band. The staining process may cause
the gel to shrink, so a larger area must be cut to insure that all of the protein is obtained.
Bands to be cut out and eluted may be spaced very close to other potentially “contami-
nating” bands. Shrinking of the reference lane may make it difficult to accurately deter-
mine the corresponding area to cut out from an unstained, unshrunk lane. In such a case,
the unstained lane(s) can be shrunk in 10% acetic acid (without methanol and Coomassie
R-250). In this way, the reduced size of the lane containing the band(s) to be eluted will
correspond to the stained gel.
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