Protein separation by electrophoresis

SDS-PAGE Sample Preparation

Prior to loading samples on a PAGE gel, it is necessary to first add sample buffer to the proteins. The sample buffer contains the detergent and buffers needed to effectively prepare a protein sample for separation by PAGE. The exact buffer to be used depends on the sample, the gel, and the conditions that will be used. 




The Bolt™ gel tank in action

 

 

 

Setting up and running a Bolt™ Bis-Tris Plus gel

The short workflow summary in Figure 7 shows how to set up the Bolt™ gel tank and Bolt™ Bis-Tris Plus gels. Similar procedures are used for setting up and running other PAGE apparatus.

Bolt-Bis-Tris-1

1. Place the base on a flat surface, and snap the electrophoresis tank into the base.

Bolt- Bis-Tris-2

2. Place the cassette clamp(s) into the electrophoresis tank. The cassette clamps are directional, so make sure they are placed in the appropriate chamber.

Bolt-Bis-Tris-3

3. Fill the chamber(s) with buffer to just above the level of the electrode.

Bolt-BisTris-4

4. Remove the comb, and remove the tape from the bottom of the gel cassette(s). Rinse the wells with 1X running buffer,and place the cassette into the electrophoresis tank.

Bolt-Bis-Tris-5

5. Close the cassette clamp by moving the lever forward so that the gel is secured firmly in place. Make sure the wells are completely filled with buffer. Load your samples and markers.

Bolt-Bis-Tris-6

6. Place the cover on the tank. Make sure the power supply is off, and plug the power leads into your power supply. Turn the power supply on to begin electrophoresis.

Recommended electrophoresis conditions for a variety of Life Technologies gel types.

Gel type Voltage Current* Run time
Bis-Tris gels* (see next table for more details)165 V constantStart 130 mA End:60 mA
  • 35–45 min, dependent on buffer type
  • Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.
Tris-glycine gels (SDS-PAGE)125 V constantStart: 30–40 mA
End: 8–12 mA
  • 90 min
  • Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.
Tris-glycine gels (native PAGE)125 V constantStart: 6–12 mA
End: 3–6 mA
1–12 hr
Tricine gels125 V constantStart: 80 mA
End: 40 mA
  • 90 min
  • Run the gel until the phenol red tracking dye reaches the bottom of the gel.
Zymogram gels125 V constantStart: 30–40 mA
End: 8–12 mA
  • 90 min
  • Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.
IEF gels100 V constant: 1 hr
200 V constant: 1 hr
500 V constant: 30 min
Start: 5 mA
End: 6 mA
2.5 hr
TBE gels200 V constant**Start: 10–18 mA
End: 4–6 mA
  • 30–90 min, dependent on gel type
  • Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.
6% TBE-urea gels180 V constant**Start: 19 mA
End: 14 mA
  • 50 min
  • Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.
10% TBE-urea gels180 V constant**Start: 15 mA
End: 8 mA
  • 60 min
  • Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.
15% TBE-urea gels180 V constant**Start: 13 mA
End: 6 mA
  • 75 min
  • Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.
DNA retardation gels100 V constantStart: 12–15 mA
End: 6–15 mA
  • • 90 min
  • Run the gel until the bromophenol blue tracking dye reaches
Table 1


* Expected start and end current values are for single gels.
** Voltages up to 250 V may be used to reduce the run time.

Recommended electrophoresis conditions for NuPAGE® Novex® Bis-Tris and Tris-acetate gels.

Gel type Voltage Expected Current* Run time
NuPAGE® Novex® Bis-Tris gels with
MES SDS running buffer
200 V constantStart: 110–125 mA/gel
End: 70–80 mA/gel
35 min
NuPAGE® Novex® Bis-Tris gels with
MOPS SDS running buffer
200 V constantStart: 100–115 mA/gel
End: 60–70 mA/gel
50 min
NuPAGE® Novex® Tris-acetate gels150 V constantStart: 40–55 mA/gel
End: 25–40 mA/gel
1 hr
NuPAGE® Novex® Tris-acetate
native gels
150 V constantStart: 18 mA/gel
End: 7 mA/gel

~2 hr
(run times may vary)
Table 2

 

† Recommended voltage for 9- and 17-well gels is 150–175 V..

Electrophoresis conditions for Bolt™ mini gels. We recommend running Bolt™ mini gels at constant voltage (1 or 2 mini gels).

Running
buffer
Expected Current* Run time* Recommended
voltage, fast run
Expected current Run
time*

MES
130 mA to 60 mA35 min 200 V180 mA to 90 mA22 min
MOPS125 mA to 40 mA45 min 200 V160 mA to 60 mA32 min

 

Table 3

 

* Run time may vary depending on the gel type and power supply used for electrophoresis


NuPAGE® Novex® gel system demonstration


 



Novex® Bis-Tris gel system demonstration