3.1 Prepare the gel sandwich

        Both prepast and self-cast gels can be run in the SE 250 or SE 260 Mighty Small II units.  Both units run gels in 10x8 cm plates, which can be cast in a Hoefer SE 215, SE 245 or SE 275 Mighty Small Gel Caster.  The SE 260 model can also accommodate longer gels in 10x10.5 cm plates, which can be cast in the Hoefer  SE 235 Mighty Small 4-Gel Caster.

        Each unit includes notched alumina plates and rectangular glass plates.  If casting your own polyacrylamide gels, we recommend using a notched alumina ceramic back plate because it transfers heat 40 times more rapidly than glass.  For applications that are not heat sensitive, a notched glass plate is available.

        Before loading gels into the electrophoresis unit, the separating gel should already be completely polymerized.  Clean away any gel adhering to the alumina back plate.  The stacking gel (if applicable) can be cast in place on the electrophoresis unit.  Load liquid samples after the gel sandwich is installed.

Note: Inspect glass plates for chipped edges. Use only unchipped plates to prevent leaking.
 

3.2 Prepare the unit

1. To disassemble a fully assembled unit: Remove the safety lid by pressing on the handle at the top of the upper buffer chamber core while lifting the lid by the bottom edges.  Empty all buffer chambers and remove any gel sandwiches.  Then depress both release tabs and life the upper buffer chamber core.

2. Rinse the instrument before each use.  Before using the first time, disassemble the unit completely and wash with dilute solution of a laboratory detergent and thoroughly rinse with water and distilled water.

3. Check the gasket.  Periodically remove the gray silicone rubber gasket from the core.  Inspect for nicks and wear.  If the gasket appears to be intact, apply a light film of Cello-Seal, and replace it in the groove.  Avoid stretching the gasket by laying it onto the groove and pressing it into place.

4. Optional cooling.
Important: Use only water or water and 50% ethylene glycol as a coolant.  Do not use a commercial antifreeze or any alcohol-based mixture.

            Circulating pressure must not exceed 0.8 bar (12 psi) above ambient pressure.  Do not connect the cooling core to an unregulated coolant source such as a water tap.

            Connect the cooling core to a circular bath such as the MultiTemp III.  Slide hose clamps (4 total) onto each end of two lengths of 8 mm (5/16”) vinyl or silicone tubing.  Attach one end of each length of tubing to a cooling core port.  Attach the free ends of each length of tubing to the circulator bath ports; one to the inlet and the other to the outlet.  Secure the connections with the hose clamps.

5. Install the upper buffer chamber core.  First steady the lower chamber with one hand and then hold the core with the other hand, position it on the positioning tabs, and press, listening for the core to snap into place. Alternatively, depress both release tabs at either side, position the core on the positioning tabs, press into place, and release the tabs.  Check that the core is secure.)

Note: If the cooling option is used frequently, it is convenient to attach QuickFit connectors to the tubing.   The valves in these fittings prevent coolant spillage.
 

3.3 Place the gel sandwich

SE 250

1. Rinse the sandwich top with distilled water to remove the overlay and drain.

2. Self-cast or pre-cast 10x8 cm plates.  Orient the sandwich so that the notched alumina plate faces the gasket and the notches are at the top.  Set the bottom of the sandwich on the bottom of the lower buffer chamber and the center the plate so that the gasket seals both sides.

SE 260

1. Rinse away the overlay with distilled water and drain any excess water.

2. If installing a self-cast or precast 10x8 cm gel sandwich, align the bottom of the plate with the bottom of the core.  The bottom of the notched plate must cover the silicone rubber gasket.

Clamp the sandwich in place

1. Lightly press the sandwich against the gasket and secure it to the core with one spring clamp on each side.  Position the jaw so that the shorter rounder jaw edge fits into the core groove and the longer edge fits on the glass plate.  (Proper positioning is important to achieve a seal and to minimize glass breakage.)  Slide the clamps down to the stop.

2. Repeat step 1 for the second sandwich, or, if running only one gel, clamp a blank cassette or a plain glass plate on the unused side of the core to prevent a possible short circuit with the unused electrode. (Do not fill this chamber with buffer if no gel sandwich is in place.)
 

3.4 Sample preparation and loading

1. If wells are already in place, skip to step 2.

If applicable, cast the stacking gel in the unit.

            Calculate the stacking gel monomer solution volume: measure the distance, in cm, from the top of the resolving gel to the notch in the alumina plate.  (This should be at least 2 cm---more if the sample depth in the well is unusually high.)  Multiply this distance by the gel width (8.3 cm) and the gel thickness (cm).  This product is the required volume in ml.

            Deaerate the stacking gel monomer solution, add catalyst and initiator and then pour.  Use a pipette to deliver the solution into one corner of the plate, taking care not to trap any bubbles.  Insert a comb (at a slight angle to prevent trapping air) into the sandwich, allowing the comb sides to rest on the spacers.

            Overlay each gel with a thin layer of water-saturated n-butanol, water, or diluted gel buffer to prevent gel exposure to oxygen.  Slowly deliver the overlay solution from a glass syringe fitted with a 22-gauge needle.  Apply the solution near the spacer at the side of the sandwich and allow it to flow across the surface unaided.  Allow a minimum of one hour for the gel to polymerize.

2. Prepare the sample.  Increase liquid sample density with 10% glycerol or sucrose.  Add a tracking dye such as phenol red or bromophenol blue.

            For SDS protein gels, use 2x treatment buffer to denature both liquid and dry samples in a test tube.  To liquid protein solutions, add an equal volume of 2x buffer.  To dry protein samples, add equal volumes of buffer and ddH20 to achieve the desired concentration.  Heat the tube in boiling water for 90 seconds, then chill it in ice until ready to use.  Treated samples can be stored frozen for future runs.  (Store at -40°C to -80°C.)

3. To aid in loading samples, wet the well-locating decal and apply it to the front of the glass plate so that the appropriate edge outlines the sample wells.

Note: The side wells for standards of a preparative comb correspond to the outermost wells formed by the 10-well comb.

Note:  Stacking gel resolution is optimal when poured just before electrophoresis. Before pouring, move the clamps to the top of the gel sandwich to prevent leaking.

Note: For dilute samples, use 6x treatment buffer as described in Appendix A.

4. Fill the sample wells and each upper buffer chamber that will be used with running buffer.  One upper buffer chamber holds approximately 75 ml.

5. Underlay the sample into the wells using a fine-tipped microsyringe.  The width of the wells depends on the number of wells per comb.  If the comb has fewer wells, they are wider, and require more volume to raise the level 1 mm, as shown:
 
 

Note: The amount of protein sample added to each well depends on both the sensitivity of the staining method and the distribution of protein among separate bands. With Coomassie Blue, it is possible to detect 1ug in a single band; with the more sensitive silver stains, it is possible to detect as little as 10 ng.
 

3.5 Final assembly

1. Fill the lower buffer chamber with running buffer.  The SE 250 lower buffer chamber holds about 150 ml and the SE 260 holds about 250 ml.  Check that the lower electrode (running along the bottom of the upper buffer chamber core) is completely submerged.

Note: If using Novex gels, check that the gel-contact slot is exposed ( the colored plastic tape must be removed).

2. Place the safety lid on the unit.

3. Plug the color-coded leads into the jacks of an approved power supply such as the Hoefer SX 250 or EPS 2A200.  The red lead plugs into the red output jack, and the black lead plugs into the black output jack.

4. OPtional cooling: Begin circulating cold water or a chilled 50/50 water/ethylene glycol solution.

Important: Do not use antifreeze or any alcohol-based mixture, as these will irreparably damage the core.
 

3.6 Running the Gel

            Precast gels are run under the same current and voltage conditions as self-cast gels.  Gels may be run at either constant current or constant voltage.  A constant current setting is traditionally used with a discontinyous buffer system so that the rate of electrophoretic migration remains unchanged throughout the run.  Under these conditions, voltage increases as the run proceeds.  A lower current setting is recommended for higher resolution.

            It takes about one hour to run two 7 cm x0.75 mm Laemmli gels at 40 mA (20mA per gel, constant current).  Check band progress after 5 minutes, and again after half an hour, keeping an eye in the position of the tracking dye.  The run is complete when the tracking dye reaches the bottom of the gel. Watch the buffer level and, if necessary, replenish it before it falls below the level of the notched plate. (A small volume of buffer may leak past a chipped plate or nicked gasket, or it may wick out through the gel.)

Important: After initial monitoring, do not leave the unit unattended for more than 45 minutes before checking the progress of the bands and the buffer level.
 

After the run

1. Once the tracking dye reaches the bottom of the gel, turn off the power supply, disconnect the leads, and remove the safety lid.

2. If coolant is circulating, stop the flow and disconnect the fittings or tubing.

3. Pour out the buffer by inverting the core assembly, then remove both clamps, and lift away gel sandwich(es) from the upper buffer chamber core.

4. Gently loosen and then slide away both spacers.  Slip an extra spacer or a Hoefer WonderWedge into the bottom edge (to prevent breaking the ears of the notched plates) and separate the plates.  The gel usually adheres to the alumina plate.  Carefully lift the gel from the plate and lay it into a tray of stain or fixative.
 

4.  Care and Maintenance

• Do not autoclave or heat any part above 45 degrees.
• Do not use organic solvents, abrasives, string cleaning solutions. or strong acids or bases to clean the chambers.

            Clean glass and alumina plates and spacers with a dilute solution of a laboratory cleanser such as RBS-35, then rinse thoroughly with tap and distilled water.  Glass plates can also be treated with (but not stored in) acid cleaning solutions.
 
 
 
 
 

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References