Bio-Rad Model 422 Electro-Eluter - Manual (Page 9)

5.2 Visualizing Proteins Using Copper Chloride
An alternative method to Coomassie R-250 is the use of the new copper chloride non fix-
ative-negative stain (Bio-Rad Copper Stain, catalog number 161-0471). This rapid, non-pre-
cipitating staining procedure surpasses Coomassie Blue in sensitivity. Following a single 5
minute incubation in CuCl
2
, proteins can be quantitatively eluted from gel slices at any time.
When the gel slice is obtained, proceed with the elution. See Section 6. [Copper Chloride: A
Five Minute Protein Stain for Sodium Dodecyl Sulfate-Polyacrylamide Gels,
Anal. Biochem.,
166, 308-312 (1987).]
5.3 Visualizing Proteins Using Autoradiography
In this method, radioactively labeled proteins are used as the reference. An aliquot of the
sample is labeled and equal amounts of labeled and unlabeled protein are loaded on separate
lanes of an SDS-polyacrylamide gel. Using the labeled protein lane as a reference, the unla-
beled protein of interest can be identified.
1. Load the labeled and unlabeled protein next to each other on the gel. Run the gel at the
optimal conditions for the band of interest. Both lanes should be run in the middle of the
gel because the end lanes may have edge effects that distort the banding pattern. Do not
allow the dye front to run off the gel.
2. After the run, cut a corner off the gel to maintain correct orientation.
3. Cut the reference lane off and place it on filter paper. Using radioactive ink (ink with
some extra isotope in it), label the top and bottom of the filter paper with the reference lane.
Dry the reference lane. Keep the rest of the gel moist by wrapping it in plastic wrap.
4. Place the dried gel in a cassette with X-ray film and expose overnight at -70 °C. After
the film has been developed, align the film and the dried lane from the gel using the marks
made with the radioactive ink. Using a lab marker, draw a line on the dried reference lane
indicating where the band is on the autoradiogram.
5. Put the rest of the gel on a glass plate. Align the gel using the cut-off corner. Put the glass
plate with the gel on top of the dried reference lane. Line up the top and bottom of the dried
lane and the gel. Using the mark made on the dried lane, cut out the appropriate area of
the gel. The dye front can be used to identify the lanes. Cut liberally to insure that all the
protein is excised. Protein diffusion should not be a problem if the band is excised the
next day.
6. After the gel slice has been cut out, proceed with the elution as described in Section 6.
Section 6
Protein Elution
6.1 Elution from SDS-PAGE Gels
1. Soak the membrane caps in protein elution buffer for at least 1 hour at 60 °C. See Section
9 for buffer recipes. Membrane caps may be soaked for longer than 1 hour. Wear gloves
when handling the Membrane Caps to prevent the dialysis membrane from becoming
contaminated.
2. Load the gel slice into the Model 422 Electro-Eluter as described in Section 4.
3. Elute at 8-10 mA/glass tube constant current for 3 to 5 hours. Elution times may vary
depending on gel percentage and the molecular weight of the protein.
6