Team: University of Chicago/Notebook/SDS-PAGE

From 2008.igem.org

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SDS-PAGE Protocols
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SDS-PAGE Protocol
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Note: Harper and Chris have gels for us. We do not need to pour any gels! Thus, acquire the gels, and begin procedures thus:
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Materials
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1. Prepare samples by heating them to 100C for 3 minutes in a 1 X SDS gel-loading buffer to denature the proteins
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polyacrylamide gel
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2. Add Tris-Glycine buffer to the top and bottom reservoirs of the apparatus.
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1X SDS buffer (6.05g Tris, 28.83g glycine, 2g SDS per liter)
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3. Load up to 15 uL of each of the samples in a predetermined order into the bottom of the wells. Wash loading device between uses. Load an equal volume of 1XSDS gel-loading buffer into any unused wells.
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Benchmark Protein Ladder
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4. Attach apparatus to power supply (+ electrode to bottom of apparatus). Apply voltage of 8 V/cm to the gel. After the dye front has moved into the resolving gel, increase voltage to 15V/cm and run gel until bromophenol blue reaches the bottom of the gel. Turn off power supply.
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Denatured protein samples
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5. Remove glass plates, place on paper towel. Use a spatula to pry plates apart. Mark the orientation of the gel by cutting a small corner from the bottom left.  
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Commassie blue (50% methanol, 10% acetic acid, 0.2% commassie)
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6. The gel can now be fixed, stained with coomassie, silver salts, or used to establish a western blot.
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Destain solution (10% methanol, 10% acetic acid)
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Procedures
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Buffers:
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1. Take the comb out of the prepared polyacrylamide gel. Place the gel in a holder and attach the holder to the PAGE apparatus.
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1XSDS gel-loading buffer
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2. Insert the apparatus in the tank and pour 1X SDS buffer in the tank and inside the apparatus.
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3. Pipette 10ul of Benchmark ladder in one lane of the gel
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50 mM Tris-Cl (pH 6.8)
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4. Pipette desired amounts of protein samples in other lanes of the gel.
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100 mM dithiothreitol
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2% SDS (electrophoresis grade)
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.1% bromophenol blue
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10% glycerol
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Tris-glycine electrophoresis buffer
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5. Put the lid on and connect the cables to the power supply
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25 mM Tris
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250 mM glycine (electrophoresis grade, pH 8.3)
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6. Set the power supply to 80V. Run for 20 minutes or until the dye front passes the stack.
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.1% SDS
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7. Increase the voltage to 120V and continue running until the dye front reaches the end of the gel.
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8. Cut off the power supply and remove gel from the apparatus.
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9. Stain gel in commasie blue stain for 1h
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10. Destain in destain solution for 30min.
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<<Protocol acquired from https://2007.igem.org/Melbourne/SDS_PAGE>>

Latest revision as of 21:09, 12 September 2008

SDS-PAGE Protocol

Materials

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)

Procedures

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.

<<Protocol acquired from https://2007.igem.org/Melbourne/SDS_PAGE>>

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