Immobiline drystrip cover fluid

Acrylamide and bis-Acrylamide were from SERVA (Heidelberg, Germany). Immobiline DryStrips, Pharmalyte buffer (broad pH range 3–10), Immobiline DryStrip cover fluid and Amersham^™ CyDye DIGE Fluors (minimal dyes) for 2D-DIGE were purchased from GE Healthcare (Piscataway Township, NJ). Complete protease inhibitor cocktail was from Roche Diagnostic (Mannheim, Germany). All others chemicals were of the highest purity available and purchased from Sigma-Aldrich (St. Louis, USA).

A PCR master mix (DimerEraser, Takara, Shiga, Japan) containing all needed components for the PCR reaction except for the primers was used. The reaction mixture was compiled with 50 µL PCR master mix, 3 µL of every primer (300 nM final concentration), 20 µL of functionalized Au NPs (1 nmol/L in distilled nuclease free H₂O), 4 µL MgCl₂ (50 mM), 4 µL NaCl (400 mM), 6 µL distilled H₂O and 10 µL target DNA, unless stated otherwise. The reaction mixture was covered by a layer of mineral oil (Immobiline DryStrip Cover Fluid, GE healthcare, Diegem, Belgium) to protect it from evaporation during thermocycling. The FO-PCR-MA was optimized for standard cycling conditions, first with an enzyme activation step at 95 °C (30 s), followed by 40 cycles of 30 s annealing at 53 °C (ramp speed = 5.0 °C/s), 30 sec elongation at 72 °C (ramp speed = 5.0 °C/s), and finally 5 s denaturation at 90 °C (ramp speed = 1.0 °C/s).

Acrylamide and bis-Acrylamide were from SERVA (Heidelberg, Germany). Immobiline DryStrips, Pharmalyte buffer (broad pH range 3–10) and Immobiline DryStrip cover fluid were purchased from GE Healthcare (Piscataway Township, NJ). Complete protease inhibitor cocktail was from Roche Diagnostic (Mannheim, Germany), [³H]-ouabain (30 mCi/mmol, NET211001MC) from Perkin Elmer and N-glycosidase F (11 365 169 001, Roche) was from Sigma-Aldrich. All others chemicals were of highest purity available and purchased from Sigma-Aldrich (St. Louis, USA).