| Procedure
Fig 2.1. SE 600 Vertical Slab Gel Unit. | Prepare the separating gel
1. Assemble the SE 600 vertical slab gel unit (Fig 2.1) in the dual-gel casting stand. Use 1.5 mm or 0.75 mm spacers (Fig 2.2a–d).
2. In a 125 ml side-arm vacuum flask, mix either 40 ml (0.75 mm) or 80 ml (1.5 mm) of resolving gel solution (Table 2.4), leaving out the ammonium persulphate and the TEMED. See Table 2.6 for recommended acrylamide concentrations.
3. Stopper the flask and apply a water vacuum for several minutes to deaerate the solution while swirling the flask (Fig 2.3).
4. Add the TEMED and ammonium persulphate and gently swirl the flask to mix, being careful not to generate bubbles.
5. Pipette the solution down the spacer into each sandwich to a level about 4 cm from the top. A 25 ml pipette works well for this step.
6. Fill a transfer pipette or syringe with water-saturated n-butanol (or water or resolving gel overlay). Position the pipette or needle at about a 45° angle with the point at the top of the acrylamide next to a spacer. Gently apply approximately 0.3 ml of n-butanol. Repeat on the other side of the slab next to the other spacer. The n-butanol will layer evenly across the entire surface after a minute or two. Repeat this process to overlay the second slab. A very sharp liquid-gel interface will be visible when the gel has polymerized (Fig 2.4). This should be visible within 10–20 min. The gel should be fully polymerized in about 1 h.
7. After polymerization, tilt the casting stand to pour off the overlay and rinse the surfaces of the gels twice with resolving gel overlay. Gels may be stored at this point. The stacking gel is cast later. Remove the n-butanol and add approximately 10 ml of 1x resolving gel overlay solution to the top of each sandwich, seal with plastic wrap, and store flat at 4 °C. Or store gels submerged flat in 1x resolving gel overlay at 4 °C for up to 1 wk.
8. Add approximately 1 ml of resolving gel overlay to each gel and allow the gels to sit while preparing the stacking gel.
Prepare the stacking gel
9. In a 50 ml side-arm vacuum flask, mix 10 ml (for 0.75-mm-thick gels) or 20 ml (for 1.5-mm-thick gels) of stacking gel solution (Table 2.4), leaving out the ammonium persulphate and the TEMED. |
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Fig 2.2a Inserting the spacer. |
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Fig 2.2b. Attaching the clamp. |
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Fig 2.2c. Properly assembled gel sandwich. Glass plates and spacer are flush. |
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Fig 2.2d. Assembling the gel casting stand (step 1). The black cams are turned to seal the bottom surface of the sandwich into the casting stand gasket. |
Table 2.4 . Resolving gel and stacking gel recipes for 1.5- and 0.75-mm-thick gels
| Resolving gel solution (80 ml; 2 ea. 1.5-mm-thick SE 600/SE 400 gels) |
| Final gel Concentration | |
| | 5% | 7.5% | 10% | 12.5% | 15% |
| Acrylamide solution | 13.3 ml | 20 ml | 26.7 ml | 33.3 ml | 40 ml |
| 4x Resolving gel buffer | 20 ml | 20 ml | 20 ml | 20 ml | 20 ml |
| 10% SDS | 0.8 ml | 0.8 ml | 0.8 ml | 0.8 ml | 0.8 ml |
| ddH2O | 45.5 ml | 38.8 ml | 32.1 ml | 25.5 ml | 18.8 ml |
| 10% Ammonium persulphate* | 400 µl | 400 µl | 400 µl | 400 µl | 400 µl |
| TEMED* | 27 µl | 27 µl | 27 µl | 27 µl | 27 µl |
| *Added after deaeration (step 3). |
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| Resolving gel solution (40 ml; 2 ea. 0.75-mm-thick SE 600/SE 400 gels) |
| Final gel Concentration | |
| | 5% | 7.5% | 10% | 12.5% | 15% |
| Acrylamide solution | 6.7 ml | 10 ml | 13.3 ml | 16.7 ml | 20 ml |
| 4x Resolving gel buffer | 10 ml | 10 ml | 10 ml | 10 ml | 10 ml |
| 10% SDS | 0.4 ml | 0.4 ml | 0.4 ml | 0.4 ml | 0.4 ml |
| ddH2O | 22.7 ml | 19.4 ml | 16.1 ml | 12.8 ml | 9.5 ml |
| 10% Ammonium persulphate* | 200 µl | 200 µl | 200 µl | 200 µl | 200 µl |
| TEMED* | 13.3 µl | 13.3 µl | 13.3 µl | 13.3 µl | 13.3 µl |
| *Added after deaeration (step 3). |
| Stacking gel solutions (4% acrylamide; for two gels) |
| Gel thickness: | 0.75 mm (10 ml total volume) | 1.5 mm (20 ml total volume) |
| Acrylamide solution | 1.33 ml | 2.66 ml |
| 4x Stacking gel buffer | 2.5 ml | 5 ml |
| 10% SDS | 0.1 ml | 0.2 ml |
| ddH2O | 6 ml | 12 ml |
| 10% Ammonium persulphate* | 50 µl | 100 µl |
| TEMED | 5 µl | 10 µl |
| *Added after deaeration (step 10). |
Fig 2.3. Mixing the gel solution under vacuum (step 3). A water aspirator is a convenient choice for this procedure | 10. Deaerate as in step 3.
11. Add the ammonium persulphate and TEMED. Gently swirl the flask to mix.
12. Pour off resolving gel overlay from the gel. Remove all liquid before proceeding.
13. Fill each sandwich with stacking gel solution and insert a comb into each sandwich, taking care not to trap any bubbles below the teeth of the comb (Fig 2.5). Oxygen will inhibit polymerization, and bubbles will cause a local distortion in the gel surface at the bottom of the wells.
14. Allow the gel to sit for at least 30 min. A very sharp interface will be visible when the gel has polymerized. This should be visible within 10–20 min. The gel should be fully polymerized after 1 h. In general, stacking gels should be cast just before use. The complete gel can be stored overnight at 4 °C, however, with little effect on resolution, if covered and the comb left in place.
Prepare the sample
15. Combine equal volumes of protein sample and 2x treatment buffer in a tube and place the tube in a boiling-water bath for 90 s. If using dry samples, add equal volumes of water and 2x treatment buffer and heat in a boiling-water bath for 90 s.
See ‘Troubleshooting’ section for more on sample preparation and how to ensure sharp bands. If the gels will be stained with Coomassie Blue, use a starting sample protein concentration of 10–20 mg/ml (i.e. 10–20 µg/µl). This will be diluted by the 2x treatment buffer to give 5–10 µg/µl. For complex mixtures (e.g. cell lysates), 50 µg of protein (5–10 µl of treated sample) per lane is recommended. For highly purified proteins, 0.5–5 µg per lane is usually adequate. Silver staining requires 10- to 100-fold less protein per lane.
16. Place samples briefly on ice until ready for use. The treated sample can be stored at 20 °C for 6 mo for future runs.
| Note: | The SDS may precipitate if tubes are left on ice for long periods of time. |
Load the gels
17. Slowly remove the combs from the gels, raising the comb up to avoid disturbing the well dividers (Fig 2.6).
18. Rinse each well with tank buffer, invert the casting stand to drain the wells, and return the stand to an upright position.
19. Fill each well with tank buffer.
20. Using a pipettor with a long, thin tip (or a Hamilton ™ syringe), gently load 5–10 µl of sample beneath the buffer in each well (Fig 2.7a). Load every well with the same volume of sample. If the well is not needed, load with 1x sample buffer containing standard protein or no sample. |
Fig 2.4. Polymerized resolving gel with n-butanol overlay. |
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Fig 2.5. Inserting comb into stacking gel (step 13). Insert the comb at an angle to avoid trapping bubbles under the comb teeth. |
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Fig 2.6. Removing comb from stacking gel (step 17). Do this gently to avoid damaging the well arms. |
This procedure ensures that each well behaves the same during separation. If an adjacent well is left empty, the adjoining samples will tend to spread laterally during electrophoresis. When adding the sample, be careful to maintain a sharp interface between the sample and the tank buffer (Fig 2.7a). Adding the sample too fast or erratically will lead to swirling and a diffuse loading zone. This will cause a loss of band sharpness (Fig 2.7b). Alternatively, the sample can be added and then overlaid with tank buffer. This is more time-consuming but, when performed carefully, minimizes contamination between wells and is particularly useful with radiolabelled samples.
21. If protein molecular weight standards are used, load one or two wells with 5–10 µl of the standard mixture. If the gel is to be stained with Coomassie™ Blue this volume should contain 0.2–1 µg of each standard component. If the gel is to be silver stained, use 10–50 ng of each component.
Run the gel
22. Fill the lower buffer chamber with 4 l of tank buffer. Install the sealing gaskets on the upper buffer chamber and put it in place on the gel sandwiches. Remove the lower cams and cam the sandwiches to the bottom of the upper buffer chamber. Put the upper buffer chamber in place on the heat exchanger in the lower buffer chamber (Fig 2.8).
23. Adjust the height of the tank buffer in the lower buffer chamber until the sandwiches are fully immersed in buffer. If bubbles are trapped under the ends of the sandwiches, coax them away with a pipette.
24. Add a spin bar to the lower buffer chamber and center the chamber on a magnetic stirrer. When the lower buffer is stirred, the temperature of the buffer remains uniform. This is important because uneven heating distorts the band pattern of the gel and leads to smiling.
25. Carefully fill the upper buffer chamber with tank buffer. Do not pour buffer into the sample wells, because it will wash the sample out.
26. Put the lid on the gel unit and connect it to the EPS 301 Power Supply. The cathode (black lead) is connected to the upper buffer chamber (Fig 2.9).
27. Turn on the power supply and adjust the voltage to 300 (so voltage is not limiting).
28. Adjust the current to 30 mA per 1.5-mm-thick gel and 15 mA per 0.75-mm-thick gel. Start the electrophoresis. The voltage should start at about 70–80 V, but will increase during the run. Keep a record of the voltage and current readings so that future runs can be compared and current leaks or incorrectly made buffers can be detected. Under these conditions, the gel will take approximately 3–4 h to run. If it is more convenient to run the gel for a longer period (e.g. 8 h), reduce the current to half—15 mA per 1.5-mm-thick gel; reduce the current to 7 mA per 1.5-mm-thick gel for 16 h (e.g. overnight).
29. When the tracking dye reaches the bottom of the gel, turn the power supply off and disconnect the power cables.
Fig 2.7a. Loading the sample (step 20). Use a very steady hand to load sample and maintain a sharp interface between the sample and the tank buffer. | 
Fig 2.7b. The wrong way to load the sample. The well on the left was loaded too quickly and with too much volume, creating a large diffuse sample zone, while the well bottom on the right was damaged with the needle. Note that the right well arm is not straight; with large volumes this will decrease resolution. |
Fig 2.8. Locking the upper buffer chamber in place. | 
Fig 2.9. The SE 600 fully assembled. |
| Disassemble the gel sandwiches
30. Remove the buffer and disassemble the sandwiches by gently loosening and sliding away both spacers. Slip an extra spacer or a Hoefer Wonder Wedge into the bottom edge and separate the plates. Carefully lift the gel into a tray of staining solution or fixative as outlined in the Gel Staining section. An example of a Coomassie Blue stained gel is shown in Figure 2.10. | 
Fig 2.10. Coomassie Blue stained gel. |
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