Melbourne/SDS PAGE
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*Time to complete protocol: | *Time to complete protocol: | ||
- | **Lab time: 15min | + | **Lab time: 15min, 5min, 5min |
- | **Waiting time:2h | + | **Waiting time:2h, 1h, 30min |
*Approximate cost of materials: $ | *Approximate cost of materials: $ | ||
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=====Primary & secondary Reagents Required including controls===== | =====Primary & secondary Reagents Required including controls===== | ||
*[[Melbourne/Preparing a poly-acrylamide gel|polyacrylamide gel]] | *[[Melbourne/Preparing a poly-acrylamide gel|polyacrylamide gel]] | ||
- | *1X SDS buffer | + | *1X SDS buffer (6.05g [[Melbourne/primary Tris|Tris]], 28.83g glycine, 2g SDS per liter) |
*Benchmark Protein Ladder | *Benchmark Protein Ladder | ||
*Denatured protein samples | *Denatured protein samples | ||
+ | *Commassie blue (50% methanol, 10% acetic acid, 0.2% commassie) | ||
+ | *Destain solution (10% methanol, 10% acetic acid) | ||
=====Method including controls===== | =====Method including controls===== |
Latest revision as of 12:35, 29 September 2007
<Return to list of protocols> <Team home page>
- 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 |
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
- Take the comb out of the prepared polyacrylamide gel. Place the gel in a holder and attach the holder to the PAGE apparatus.
- Insert the apparatus in the tank and pour 1X SDS buffer in the tank and inside the apparatus.
- Pipette 10ul of Benchmark ladder in one lane of the gel
- Pipette desired amounts of protein samples in other lanes of the gel.
- Put the lid on and connect the cables to the power supply
- Set the power supply to 80V. Run for 20 minutes or until the dye front passes the stack.
- Increase the voltage to 120V and continue running until the dye front reaches the end of the gel.
- Cut off the power supply and remove gel from the apparatus.
- Stain gel in commasie blue stain for 1h
- Destain in destain solution for 30min.
Equipement Required
- Pipette and tips
- SDS PAGE apparatus
- Power source