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Team: University of Chicago/Notebook/SDS-PAGE
From 2008.igem.org
SDS-PAGE Protocols
Note: Harper and Chris have gels for us. We do not need to pour any gels! Thus, acquire the gels, and begin procedures thus:
1. Prepare samples by heating them to 100C for 3 minutes in a 1 X SDS gel-loading buffer to denature the proteins
2. Add Tris-Glycine buffer to the top and bottom reservoirs of the apparatus.
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.
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.
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.
6. The gel can now be fixed, stained with coomassie, silver salts, or used to establish a western blot.
Buffers:
1XSDS gel-loading buffer
50 mM Tris-Cl (pH 6.8) 100 mM dithiothreitol 2% SDS (electrophoresis grade) .1% bromophenol blue 10% glycerol
Tris-glycine electrophoresis buffer 25 mM Tris 250 mM glycine (electrophoresis grade, pH 8.3) .1% SDS
