Amersham prime western blotting detection reagent

Tumor cells (5×10⁵) were lysed with RIPA buffer (Tris-buffered saline containing 1 % Nonidet P-40, 0.5 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate, 0.0004 % sodium azide, 0.2 M orthovanadate and 0.5 M phenylmethyl-sulfonyl fluoride). Of each sample 25 μl were mixed with 5 μl 5x Laemmli-buffer and applied to a SDS-Gel (9 %). Following blotting, the membrane was incubated in 5 % milk powder in TBS, containing 0.1 % Tween20. Of the antibody to T2R38 (sc-34294, Santa Cruz, Heidelberg, Germany) a 1:1000 dilution was used, of antigen peptide sc-34294P 0.5 μl/10 μl antibody. Secondary antibody was a POX conjugated mouse anti-goat IgG (Jackson Immuno Research, Suffolk, UK, 1:20000). Blots were developed using Amersham Prime Western Blotting detection Reagent (GE Healthcare, Freiburg, Germany).

Cells (1 × 10⁷) were lysed with RIPA buffer (Tris-buffered saline containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.0004% sodium azide, 0.2 M orthovanadate and 0.5 M phenylmethyl-sulfonyl fluoride) and incubated overnight at 4°C with or without AHL-12-FITC (300 μM; Cayman Chemical), then with sepharose beads (Cell Signaling, Danvers, MA, USA). The supernatant was subsequently incubated with an antibody to FITC, coupled to sepharose (2 h at 4°C). Adsorbed fractions were eluted with SDS loading buffer containing β-mercaptoethanol. The samples were applied to a 12% SDS-Gel. Silver staining was performed and western blotting using anti-T2R38 (sc-34294) or an anti-IQGAP (abcam). Secondary antibodies were a POX-conjugated mouse anti-goat IgG (#205-035-108, Jackson Immuno Research, Suffolk, UK, 1:20000). Blots were developed using Amersham Prime Western Blotting Detection Reagent (GE Healthcare, Freiburg, Germany).