User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/04/15

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SDS Page Gel Electrophoresis I. 4 - 12 well pre-cast Mini Protean TGX gel was obtained and prepared. The comb and tape was removed from each gel The wells of each gel were rinsed with the prepared running buffer dilution II. The Bio-Rad Mini Protean system electrophoresis cell was assembled. The inner and outer buffer chambers were filled with the diluted running buffer to the "4 gel" mark. III. Each of the 4 gels were loaded with each of the 20 μL reaction samples prepared yesterday in the following manner: The samples were heated for 5 min at 100C in the thermocycler pre-loading Gel AO Well Sample 1 Ladder 2 BLANK 3 1 μM Proteinase K (I) 4 100 nM Proteinase K (I) 5 10 nM Proteinase K (I) 6 1 nM Proteinase K (I) Gel AI Well Sample 1 Ladder 2 BLANK 3 1 μM Proteinase K (II) 4 BLANK 5 100 nM Proteinase K (II) 6 BLANK 7 10 nM Proteinase K (II) 8 BLANK 9 1 nM Proteinase K (II) Gel BI Well Sample 1 Ladder 2 BLANK 3 1 μM Trypsin 4 100 nM Trypsin 5 10 nM Trypsin 6 1 nM Trypsin 7 BLANK 8 1 μM Chymotrypsin 9 100 nM Chymotrypsin 10 10 nM Chymotrypsin 11 1 nM Chymotrypsin Gel BO Well Sample 1 Ladder 2 BLANK 3 1 μM Thermolysin 4 SKIP 5 100 nM Thermolysin 6 SKIP 7 10 nM Thermolysin 8 BLANK 9 1 nM Thermolysin IV. The gel was run for approximately 10 min at 200 V, 0.05 A, and 10 W. NOTE: The top of the chamber was not functioning properly in that a closed circuit was not established and the top had to be manually held/pushed down for the entirety of the run. V. After 10 min, the gel was placed in a Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes VI. The gel was then placed in a Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for approximately 24 hours VII.Finally, the gel was placed in Destain Solution (10% acetic acid, 90% water) three times for 15 minutes each and the following results were obtained: