Protein Protocols Protein Protocols

DIGE 191
Strips, the instructions that come with the gels should be followed in general; however,
do note that some of the solutions and procedures recommended by Pharmacia have
been modified here. When there’s a conflict, use this version for the best results.
3.3.1. Rehydration
1. The rehydration cassette is made up of two glass plates, one with a 0.5 mm rubber
U-gasket, the other plain. Wash both plates with H2O, then 95% EtOH and air dry.
2. Hold gel strip at either end and peel off protecting thin layer of plastic. The thicker piece
has the gel cast on it. Discard the protecting plastic. The gel is bonded to special plastic
called polyacrylamide (PAG) bond plastic. The back side of the strip opposite the gel is
hydrophobic. Drop just a few drops of water on this hydrophobic side (see Note 10). Lay
the plain glass plate face down and place the gel on it, wet (hydrophobic) side down. Make
sure the acidic (pointed) end is near the bottom of plate.
3. After all gels are in place, roll them flat with a Teflon roller. There is no need to press
hard; all that is needed is that the strips don’t fall down when the glass plate is inverted.
Remove any excess water that might have seeped out.
4. Make sure gasket around the edge of top glass plate is clean and dry, then place over
bottom plate and clamp two plates together (two clips on each long side and one at bottom).
5. Using the special 25-mL syringe included with the kit, fill cassette with rehydration buffer
by injecting from the bottom until the top of the gels are just covered. Try to avoid air
bubbles. Lay the cassette assembly flat and rehydrate overnight (see Note 11).
3.3.2. Setting Up and Running the First Dimension
1. Remove the rehydration buffer from the IEF strips and save at 4°C for future reuse (see
Note 6).
2. To make the contact wicks that will go between the gel and the electrodes, cut the filter
paper supplied with the Immobiline apparatus to approx 0.5 × 0.3 mm rectangles (see
Note 12). The wicks are used for ensuring good contact between the electrodes and the
gel. They also act as a buffer space for salts that migrate to the electrodes. Cut enough
wicks so that there are a few more than twice the number of strips. Immerse all wicks in
HPLC quality H2O for about a minute. Then place all wicks on a paper towel so that they
cluster around each other to create a damp patch on the paper. They should be placed close
to but not overlapping with each other. Then start placing the IEF strips on the gel apparatus.
By the time the gels are in place and ready to receive the wicks, they are usually at the
right dampness (see Note 13).
3. Lay down a thin line of light paraffin oil into each groove on the strip aligner that will
receive an IEF strip. Make sure to use a minimal amount of oil. It is not desirable to have
any excess oil on top of the gel, especially over the location of the sample cup. Place one
IEF strip over each oil streak with the acidic end pointing toward the positive, red
electrode, and making sure that it is parallel to and aligned with the other gels using the
lines on the flatbed.
4. Apply one wick at each (basic and acidic) end of each gel. Make sure all the wicks are
lined up with each other if there is more than one IEF strip on the plate. Place both electrodes
over their respective wicks. Take care to orient the electrodes correctly. They are
color coded and the instructions are included in the kit. Wrong orientation of the electrodes
will result in no electrophoresis. Samples will be applied close to the anode—so the
sample cup holder should next be placed as close to the positive electrode as possible.
5. Cover the gel strips with Teflon membranes, except the areas where the sample cups are
going to be applied (see Note 14). Dab gel area around prospective sample cup location
with a paper towel if there’s liquid over it; otherwise leave it alone. After putting the cups