Western-Blot-Protean.doc - Melissa Landis SDS-PAGE Thaw...

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Melissa Landis a512665f03678af630457fa7d4e5aa0d64c104be.doc 4/29/19 SDS-PAGE Thaw samples on ice. Assemble gel mold Clean 1 small and 1 large plate with glass cleaner Add spacers. Add clamp to each side. align Align in holder. Pour EtOH to ensure tight seal. Remove EtOH. Pouring gel ***Polyacrylamide is a very potent neurotoxin ****wear gloves*** Mark a line about 1.5 cm below the bottom of the comb teeth. Combine following reagents: Resolving Gel (2) 8% 10% 12% 14% Stacking Gel (2) 5% ml ml ml ml ml 30% acrylamide/bis 26.5 33.5 39.5 46.5 30% acrylamide/bis 8.3 ddH20 47 40 34 27 ddH20 34 1.5M Tris, pH 8.8 25 25 25 25 0.5M Tris, pH 6.8 6.3 20%SDS 0.5 0.5 0.5 0.5 20%SDS 0.25 10% APS 1 1 1 1 APS 0.5 TOTAL 100 100 100 100 TOTAL 49.35 Add 50 l TEMED to resolving gel. Mix by pipetting. Pour to the drawn line. Add methanol to both corners. Allow to polymerize >30 min. Pour off methanol. Add 50 l TEMED to stacking gel. Pour, overfill, add combs. Polymerize 15 min. Remove combs. Wash with ddH20 bottle. Set up gel running system. Turn plates so that the combs are inside. Fill tank with running buffer.
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Melissa Landis a512665f03678af630457fa7d4e5aa0d64c104be.doc 4/29/19 Prepare samples & load samples Well number 1 2 3 4 5 6 7 8 9 10 11 12 Sample name Concen ( g/ l) Volume loaded Well number 1 2 3 4 5 6 7 8 9 10 11 12 Sample name Amt ( g) Volume loaded Boil 3 minutes (because SDS precipitates when frozen), flash spin. Thaw marker. Load samples. Load 40 l Invitrogen BenchMark protein standard. Run 35mAmps per gel (70 for 2) for ~1H until protein is through stacking gel. Run 50mAmps per gel (100 for 2). Running Buffer 100ml 10X Running Buffer 5 ml 20% SDS Up to 1L with H 2 0
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Melissa Landis a512665f03678af630457fa7d4e5aa0d64c104be.doc 4/29/19 Wet Transfer Cut 15 X 20 cm PVDF membrane. **Wear gloves** Cut top right corner off. Label membrane
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