Bio-Rad PROTEAN II XL Cell - Manual (Page 31)

25
Running the IEF Gels
1. Fill the central cooling core with water or coolant. Cap the inlet and outlet port with the caps provided.
2. Fill the lower buffer chamber with ~4.5 liters of lower running electrolyte (see Section 14). Place on a
level surface or leveling table.
3. Lower the cooling core/tube apparatus into the lower chamber of the PROTEAN ® II xi cell. Lower
carefully so as not to introduce any air bubbles under the gel tubes. All bubbles must be removed from
the bottom of the tubes to insure proper electrical contact.
4. Pour 325-350 ml freshly degassed upper running electrolyte (see Section 14) into the upper buffer
chamber, put the lid on, and attach the power cables to the power supply.
5. Run the first dimension gels at room temperature with a constant voltage of 200 volts for 2 hours,
followed by 500 volts for 2 hours, and then 800 volts overnight (16 hours). As a safety precaution,
always set voltage, current, and power limits when possible.
Note: Phycocyanin is a colored protein found in Bio-Rad’s IEF standards (catalog number 161-0310).
Although these standards cannot be used for pI calibration in 2-D procedures, because denaturation in
urea produces too many peptide spots, the phycocyanin is excellent for monitoring the first dimension
IEF. It retains its blue color and will focus in a tight blue band when focusing is finished. Loading one
tube with the focusing standards is an easy way to monitor the progress of the focusing run.
6. Cast the second dimension SDS slab gel during the running of the first dimension (see Section 4).
Note: This protocol does not use a stacking gel. However, if a stacking gel is required for a particular
application, it should be cast on a level surface. It is important that the same amount of monomer be
used for each stacking gel to insure stacking gels of identical depth. If a comb is not used, as in most
2-D applications, then the stacking gel should be overlaid with 1.0 ml of water saturated sec-butanol.
After polymerization is complete, drain off the overlay (or remove the combs), and rinse the gel surface
briefly with distilled deionized water.
Extruding Tube Gels
1. After electrophoresis, remove the tube gels from the tube gel adaptor. Rinse both the top and bottom of
each gel thoroughly with distilled water. Failure to rinse before extrusion will result in residual base
(NaOH) or acid (phosphoric) on the gel that will interfere with measurement of the pH gradient. Place the
tubes, in order, into the tube rack, and fill each tube to the top with distilled water.
2. Attach a long (at least 2 inch), fine (at least 26 gauge), beveled needle (such as catalog #165-1944) to a 3
ml plastic syringe. Rim the upper and lower few mms of the gel by inserting the needle between the gel
and the glass tube (point against the glass wall to prevent tearing of the gel) while forcing distilled water
through the syringe and needle. Turn the gel tubes so that the entire circumference is rimmed (see photo).
Often tube gels may be extruded without rimming, using water pressure as below.