Bio-Rad PROTEAN II XL Cell - Manual (Page 29)

23
1. Mark the capillary tubes (1.5 mm ID, 6.0 mm OD, 180 mm, catalog number 165-3138) with a laboratory
marker 14.0 cm from one end.
2. Connect each capillary tube to a 1 ml syringe using a small piece of Tygon tubing 3⁄16" ID x 1⁄4" OD,
and approximately 2 cm in length (not included). Fill either a test tube rack or a level casting stand, such
as Bio-Rad’s Model 225 Tube Gel Casting Stand (catalog number 165-2020) with a disposable 12 x 75
mm test tube for each capillary tube. Insert a capillary tube/syringe assembly into each test tube.
3. Prepare the first dimension monomer solution and degas well. (The removal of molecular oxygen by
degassing is essential for reproducible polymerization.)
Warning: Always wear gloves to prevent exposure to acrylamide.
4. Add the APS and TEMED, and swirl 8 to 10 times. Working quickly, pipet 1 ml of acrylamide solution into
each test tube. Using the syringe, pull up the liquid in each tube to the 14.0 cm mark. Let the capillary
tubes sit undisturbed, with syringes attached, for 2 hours at room temperature to allow complete
polymerization to occur.
5. After polymerization is completed, remove the capillary tubes from each test tube. Remove the syringe and
Tygon tubing. Press and rotate the bottom of the capillary tube squarely down on a piece of Parafilm to
remove excess acrylamide. Wipe off the excess acrylamide.
6. Inspect the gels before loading; bubbles within the gel prevent focusing and these gels should be discarded.
Note: Alternative methods for filling capillary tubes can be used, such as wrapping the bottom end of the
capillary tube with two layers of Parafilm laboratory film and filling using a syringe and fine gauge cannula
(gel tube loading needle, 165-1943). The cannula should be long enough to reach the bottom of the tube.
Slowly inject the acrylamide solution into the bottom of the tube, withdrawing the cannula as the acrylamide
enters the tube. Fill to the mark on the tube.
Sample Preparation and Loading
Sample preparation prior to isoelectric focusing is one of the most important steps for obtaining reproducible
two-dimensional electrophoresis gels. There is no method which is optimal for every sample, and it may be
necessary to experiment with different protein solubilization methods to determine which is best.
1. Prepare the first dimension running solutions as described in Section 14.
2. Prepare the IEF sample concentrate solution A and/or iso-urea solution E as described in Section 14.
These solutions should be prepared fresh, or frozen in aliquots.
Note: Sample loads above 400 µg total protein may cause loss of resolution in the second dimension
slab gel.
3. Replace the notched white gaskets with the tube adaptor gaskets specifically designed for use during
the first dimension of 2-D electrophoresis. Do not lubricate or wet the red tube adaptor gaskets.
Note: Tube gels have a much higher resistance than slab gels due to their small diameter. Since current
seeks the path of least resistance, a current leak may occur if there is an alternative path of conductance
such as a wet gasket. A current leak is a safety hazard to the researcher as well as the equipment.
4. Attach the tube gel adaptor to the cooling core in the same manner that a slab gel sandwich is attached
(see Section 5.1). Sandwich clamps are not needed to attach the tube adaptor to the core. Finish
assembling the upper buffer chamber with a second tube gel adaptor. Because of the higher voltages
required for focusing, we recommend always using two tube gel adaptors and not the buffer dam for
focusing.