Bio-Rad Model 422 Electro-Eluter - Manual (Page 14)

Section 9
Recipes
1. Protein Elution Buffer
Tris base 3.0 g 25 mM
Glycine 14.4 g 192 mM
SDS 1.0 g 0.1%
to 1 liter with dH
2
0
Store at 4 °C. Warm to 37 °C before use if precipitation occurs.
2. Volatile Buffer (for protein elution and concentration)
Ammonium bicarbonate (NH
4
HCO
3
) 3.95 g 50 mM
SDS 1.0 g 0.1%
to 1 liter with dH
2
0
Make up only 1 liter at a time because the buffer will volatilize. Store at 4° C.
3. DNA Elution Buffer (50x) Working Concentration
Tris 242 . g 40 . mM
Glacial acetic acid 57.1 ml 20 . mM
0.5 M EDTA (pH 8.0) 100 . ml 1 . mM
SDS 5 . g 0.1%
to 1 liter with dH
2
0
Section 10
Troubleshooting Guide
SDS is included in elution buffer recipes to insure that the net negative charge of eluted
molecules is maintained. As a result, the eluted molecules migrate toward the anode (positive
electrode) and end up in the elution cup, SDS concentrates in the lower buffer chamber. If high
SDS concentration is undesirable, at the end of the run the lower buffer can be exchanged
for fresh elution buffer made without SDS, and the run continued for approximately 1/2 hour.
This will effectively electrodialyze SDS from the collected sample.
Proteins originally separated in native gels can be eluted without inclusion of SDS in the
elution buffer. Because elution without SDS requires an approximately 25% increase in run
times, and the volume of buffer contained in the Model 422 Electro-Eluter is small relative to
a standard vertical slab electrophoresis apparatus, the buffer may require replenishment dur-
ing these extended runs (very high MW proteins especially) to avoid “buffer depletion.”
Buffer depletion is also problematic when running the eluter with more than the recom-
mended 1 cm of gel per elution tube, which may require runs exceeding 6-7 hours. Even in
the presence of SDS, extended runs (6 hours to overnight) will require at least one change of
upper and lower elution buffer. The buffering capacity of the elution buffer maintains the pH
at which molecules maintain electrophoretic mobility. Alkaline pH (8-9) is standard for most
Tris-glycine or phosphate based native electrophoresis/elution buffers.
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