Bio-Rad Model 422 Electro-Eluter - Manual (Page 10)

4. After the elution is completed, remove the electro-eluter module from the buffer tank.
Wear gloves to prevent contamination of the dialysis membrane.
5. Take the module, with the Glass Tubes attached, to a sink. If a stopper has been used,
remove the stopper from the upper buffer chamber and allow the buffer to drain out. If
there are no stoppers, carefully pull one glass tube out of the upper buffer chamber and
allow the upper buffer to drain.
6. Using a plastic pipet, remove the buffer left in the tube to the level of the Frit and discard
the liquid. Work quickly and carefully. Do not dislodge the Silicone Adaptor or the mem-
brane cap. Make sure that the liquid below the Frit is not disturbed or shaken up during
this process.
7. Remove the Silicone Adaptor together with the membrane cap from the bottom of the
glass tube. The liquid level should be slightly above the membrane cap. Using a new
plastic pipet, pipet the remaining liquid in the membrane cap into a microfuge tube. The
volume should be approximately 400 µl. With another 200 µl of fresh elution buffer, rinse
the membrane cap. Add the rinse solution to the microfuge tube. This solution will con-
tain the eluted protein. Do this for each Glass Tube. The yield for eluted proteins is
between 80-100%.
8. The membrane cap may be reused for at least five complete runs without decreasing the
yield. Refrigerate the membrane cap in elution buffer with 0.05% sodium azide (NaN
3
).
It is not necessary to reheat or resoak the membrane caps after the first use.
6.2 Elution of Fixed and Stained Proteins
With the Model 422 Electro-Eluter, it is possible to elute proteins that have been fixed and
stained. If the fixing and staining process will not affect subsequent experiments, this is the
preferred method. However, yields may be decreased slightly (10-20%) due to precipitation
of proteins by methanol and/or cross-linking of the molecules by the dyes or stains. If bands
are too closely spaced to allow estimation of their location in the gel by extrapolating from a
stained reference lane (see Section 5.3), the following method should be followed to elute
fixed and stained proteins.
1. Visualize the protein by staining the gel with Coomassie R-250 as described in Section
5.1.
2. Cut out the band of interest. Elute the proteins following the protocols in Section 6.
3. The yield of fixed and stained proteins is from 70% to 90%. Some protein may remain in
the gel slice.
6.3 Concentration of Eluted Proteins Using Volatile Buffers
1. The Model 422 Electro-Eluter is assembled as described in Section 4. The proteins are
visualized using one of the methods described in Section 5.1 or 5.2.
2. The protein is eluted from the gel as described in Section 6, except a volatile elution
buffer is used. See Section 9 for recipes for the volatile buffer.
3. After the elution, the volatile buffer is lyophilized in a spin-vacuum, leaving concentrat-
ed protein. Elution yields using the volatile buffer are from 80% to 100%.
6.4 Elution of Proteins from Native Gels
Native proteins separated on non-denaturing slab gels can be eluted using the same native
gel buffer system employed during electrophoresis in the slab (e.g. Ornstein-Davis buffer). 1
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