Knight:NuPAGE electrophoresis

In progress! For a better version of this protocol, see Sauer:bis-Tris SDS-PAGE, the very best.

Purpose

To run a denaturing protein gel. Note that a preferable version of this protocol is available at Sauer:bis-Tris SDS-PAGE, the very best (recommended by Kathleen). But since we seem to already have the Invitrogen kit contents in lab, this protocol describes use of the Invitrogen system.

Materials

• NuPAGE Novex 10% Bis-Tris Gel 1.0 mm, 10 well
• XCell SureLock Mini-Cell electrophoresis apparatus
• 20X MES Running Buffer
• NuPAGE LDS Sample Buffer (4X)
• NuPAGE Reducing Agent (10X)
• NuPAGE Antioxidant

Procedure

Running buffer preparation

Do this step first.

1. Prepare 1000mL 1X NuPAGE SDS running buffer
   • 50mL 20X MES Running Buffer
   • 950mL deionized water
2. Mix well.
3. Set aside 200mL buffer for use in Upper (Inner) buffer chamber).
   • Add 500μL NuPAGE Antioxidant immediately before running the gel.
   • Mix well.

Sample preparation

1. Wells can accommodate 25μL loading volume.
2. Let sample buffer warm to room temperature.
3. Each sample should contain
   • 13 μL protein sample (max 0.5μg per band)
   • 5 μL NuPAGE LDS Sample Buffer
   • 2 μL NuPAGE Reducing Agent
4. Heat samples at 70°C for 10 mins.

Set up the gel apparatus during the 10 mins sample heating step.

Note that prestained molecular weight marker doesn't need any preparation.

Running the gel

1. Wear gloves.
2. Remove the NuPAGE gel from the pouch.
3. Rinse the gel cassette with deionized water.
4. Peel the tape from the bottom of the cassette.
5. Gently pull the comb from the cassette in one smooth motion.
6. Rinse the sample wells with 1X NuPAGE SDS running buffer.
   • Use a pipetman and pipet to squirt in running buffer.
7. Invert and shake to remove buffer.
8. Repeat rinse two more times.
9. Orient the two gels in the Mini-Cell such that the notched "well" side of the cassette faces inward towards the buffer core.
10. Seat the gels on the bottom of the Mini-Cell and lock into place with the gel tension wedge.
    • Use the plastic buffer dam if you are only running one gel.
11. Fill the upper buffer chamber with a small amount of uppder buffer chamber running buffer (with antioxidant) to check tightness of seal.
    • If there is a leak, discard buffer, reseal chamber and try again.
12. Fill upper buffer chamber. Buffer level should exceed level of the wells. Requires about 200mL
13. Load samples.
14. Load protein molecular weight marker.
15. Fill lower buffer chamber at the gap near locking mechanism with 600mL NuPAGE SDS running buffer.
16. Run at 200V for 30 minutes.

Staining the gel

1. Shut off the power.
2. Disconnect electrodes.
3. Remove gels.
4. Insert a knife in between the two plates and pry the plates apart.
   • You should hear a cracking noise as you break the bond between the two plates.
5. Gently separate the two plates attempting to leave the gel on the bottom slotted plate.
6. Cut to separate gel from bottom lip.
7. Flip over and transfer gel to staining tray that has been prefilled with 100mL deionized water (see below).

You have two options for staining at this point.

Fast, microwave protocol

1. Add 100mL deionized water in staining tray.
2. Microwave staining tray loosely covered for 1 min on high.
3. Shake staining tray for 1 min on orbital shaker at room temperature.
4. Discard water.
5. Repeat steps 1-4 two more times.
6. Add 20mL SimplyBlue SafeStain.
7. Microwave staining tray loosely covered for 45 secs on high.
   • 1 min seemed a bit too long.
8. Shake staining tray for 5 mins on orbital shaker at room temperature.
9. Discard SimplyBlue SafeStain.
10. Add 100mL deionized water.
11. Shake staining tray for 10 mins on orbital shaker at room temperature.
12. Add 20mL 20% NaCl for at least 5 mins.
13. Gel can be stored in salt solution for several weeks.

This protocol seems to work pretty well.

Slow, room temperature protocol

1. Add 100mL deionized water in staining tray.
2. Shake staining tray for 5 min on orbital shaker at room temperature.
3. Discard water.
4. Repeat steps 1-3 two more times.
5. Add 20mL SimplyBlue SafeStain.
6. Shake staining tray for 1 hr on orbital shaker at room temperature.
7. Add 100mL deionized water.
8. Shake staining tray for 1 hr or more on orbital shaker at room temperature.

This protocol worked less well for me. However, I learned later that the typical trick people use is to include a kimwipe in the destain step to help soak up the stain. This will likely improve the signal to noise on the gel.

Safety

• Use nitrile gloves when handling acrylamide.
• Dispose of acrylamide gels and trays as hazardous.

See here for detailed safety information.

References

1. NuPAGE Technical Guide