Last Revision: 4 November 2017 Part II

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SDS-PAGE Procedure

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 2.2ad).
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.
Fig 2.1. SE 600 Vertical Slab Gel Unit. 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.
Fig 2.2a Inserting the 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 1020 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
Fig 2.2b. Attaching the clamp. 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.
10. Deaerate as in step 3.
11. Add the ammonium persulphate and TEMED. Gently swirl the flask to mix.
Fig 2.2c. Properly assembled gel sandwich.
12. Pour off resolving gel overlay from the gel. Remove all liquid before
Glass plates and spacer are flush.
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 1020 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

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Fig 2.2d. Assembling the gel casting stand effect on resolution, if covered and the comb left in place.
(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 l 400 l 400 l 400 l 400 l
TEMED* 27 l 27 l 27 l 27 l 27 l
*Added after deaeration (step 3).
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 l 200 l 200 l 200 l 200 l
TEMED* 13.3 l 13.3 l 13.3 l 13.3 l 13.3 l
*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 l 100 l
TEMED 5 l 10 l
*Added after deaeration (step 10).

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 1020 mg/ml (i.e. 1020 g/l). This will be diluted by
Fig 2.3. Mixing the gel solution under the 2x treatment buffer to give 510 g/l. For complex mixtures (e.g. cell
vacuum (step 3). A water aspirator is a lysates), 50 g of protein (510 l of treated sample) per lane is recommended.
convenient choice for this procedure For highly purified proteins, 0.55 g 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.

Fig 2.4. Polymerized resolving gel with n-

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butanol overlay.

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.

Fig 2.5. Inserting comb into stacking gel 20. Using a pipettor with a long, thin tip (or a Hamilton syringe), gently load
(step 13). Insert the comb at an angle to 510 l of sample beneath the buffer in each well (Fig 2.7a). Load every well with
avoid trapping bubbles under the comb the same volume of sample. If the well is not needed, load with 1x sample buffer
teeth. containing standard protein or no sample.

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 510 l of the standard mixture. If the gel is to be
stained with Coomassie Blue this volume should contain 0.21 g of each standard component. If the gel is to be silver stained,
use 1050 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 7080 V, but will increase during the run. Keep a record of the voltage and current readings so that future
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runs can be compared and current leaks or incorrectly made buffers can be detected. Under these conditions, the gel will take
approximately 34 h to run. If it is more convenient to run the gel for a longer period (e.g. 8 h), reduce the current to half15 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 Fig 2.7b. The wrong way to load the sample. The well on the left was
steady hand to load sample and maintain a sharp loaded too quickly and with too much volume, creating a large diffuse
interface between the sample and the tank buffer. 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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