Anti phospho1 recombinant fab

Cells were lysed in RIPA buffer (20 mM Tris–HCl, pH. 8.0, 135 mM NaCL, 10% glycerol, 1% IGEPAL, 0.1% SDS, 0.5% Na deoxycholate, 2 mM EDTA; Invitrogen) containing “complete” protease inhibitor cocktail according to manufacturer's instructions (Roche) and protein concentration determined using the standard DC assay (Bio-Rad, Hemel Hempsted, UK). Proteins (8 µg) were run in a 10% Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose membrane. The membrane was blocked with Odyssey blocking buffer (LI-COR Biosciences, Nebraska, USA) for 1 h and probed overnight at 4 °C with anti-PHOSPHO1 (recombinant Fab, AbD Serotec, Martinsried/Planegg, Germany) anti-TNAP (R&D, Abingdon, UK) and anti ß-actin (Cell signaling technology, Hitchin, UK) antibodies diluted 1:1000,1:500 and 1:1000 respectively in Odyssey blocking buffer. After washing in PBS the membranes were incubated with goat anti-Human (800CW), goat anti-Rat (800CW) and goat anti-Rabbit (680 RD) for 50 min (LI-COR Biosciences, 1:1250 dilution in Odyssey blocking buffer). The resulting blots were subsequently washed in PBS and visualized using the LI-COR Odyssey infrared scanner and software (LI-COR biosciences) with a scan resolution of 169 µm.