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Summary: 95
SDS-PAGE and Transfer to PVDF Membrane for Protein Sequencing
· Prepare all solutions in scrupulously clean glassware. Avoid introduction of contamination at every
step. Carefully clean glass plates, spacers and combs for the gel. Use the highest quality reagents.
1. Prepare gel solutions:
1X Upper 1X Lower 4X Gel
BisTris 11.83 g 6.55 g 51.55 g
TES 5.04 g -- --
SDS 0.5 g -- -- Use ultrapure SDS.
HCl (5.8 M) -- 3 mls 15 mls Add 5.8 M HCl slowly with stirring.
pH (25°C) 6.9 - 7.0 5.90 6.60
Final Volume 500 mls 500 mls 500 mls Syringe filter 100 mls for gels.
Keep 400 mls for electrophoresis.
2. Pour GIBCO/BRL gel as usual, but make the following changes:
10.5% Resolving 9% Resolving 4% Stacking4
4X Gel Buffer 5 mls 5 mls 1.25 mls
30% Acrylamide1 7 mls 6 mls 0.75 ml
ddH2O2 8 mls 9 mls 3 mls
TEMED3 20 µl 20 µl 5 µl
25% APS3 20 µl 20 µl 10 µl
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