From 2007.igem.org

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• Applications:
• Separation of proteins on a gel
• Western blots

• Time to complete protocol:
  • Lab time: 15min, 5min, 5min
  • Waiting time:2h, 1h, 30min
• Approximate cost of materials: $

Contents 1 Method from primary and secondary reagents 1.1 Primary & secondary Reagents Required including controls 1.2 Method including controls 1.3 Equipement Required 1.4 References

Method from primary and secondary reagents

Primary & secondary Reagents Required including controls

• polyacrylamide gel
• 1X SDS buffer (6.05g Tris, 28.83g glycine, 2g SDS per liter)
• Benchmark Protein Ladder
• Denatured protein samples
• Commassie blue (50% methanol, 10% acetic acid, 0.2% commassie)
• Destain solution (10% methanol, 10% acetic acid)

Method including controls

1. Take the comb out of the prepared polyacrylamide gel. Place the gel in a holder and attach the holder to the PAGE apparatus.
2. Insert the apparatus in the tank and pour 1X SDS buffer in the tank and inside the apparatus.
3. Pipette 10ul of Benchmark ladder in one lane of the gel
4. Pipette desired amounts of protein samples in other lanes of the gel.
5. Put the lid on and connect the cables to the power supply
6. Set the power supply to 80V. Run for 20 minutes or until the dye front passes the stack.
7. Increase the voltage to 120V and continue running until the dye front reaches the end of the gel.
8. Cut off the power supply and remove gel from the apparatus.
9. Stain gel in commasie blue stain for 1h
10. Destain in destain solution for 30min.

Equipement Required

• Pipette and tips
• SDS PAGE apparatus
• Power source

References