Irdye 680lt donkey anti mouse secondary antibody

Western blotting was performed using standard methods. Briefly, cells were scraped in PBS and lysed in RIPA buffer (150mM NaCl, 1% NP-40 alternative, 0.5% Sodium deoxycholate, 0.1% SDS, 40mM Tris pH8.0) with 1mM Benzamidine HCl, 1µg/mL antipain, 5µg/mL aprotinin, 1µg/mL leupeptin, 1mM Phenylmethansulfonyl fluoride, 10mM NaF, 2mM imidazole, 1.15mM Sodium molybdate, 4mM Sodium tartrate and 2mM Sodium orthovanadate (Sigma)) for 20 minutes on ice and cleared by centrifugation. Protein was quantified using Bradford reagent and 20–30µg of protein were run per lane of 4–12% Bis-Tris gels (Thermo Fisher Scientific) using either MOPS or MES buffer. Protein was transferred to nitrocellulose membrane at 350mA for 2h in transfer buffer (15% methanol, 191mM glycine, 25mM Tris base, 0.1% SDS, pH8.3). Membranes were blocked in 5% non-fat milk powder in PBS+0.2% Tween 20 (PBST) and incubated overnight at 4°C in primary antibody diluted in blocking solution. Primary antibodies are listed in Table 2. After extensive washing in PBST blots were incubated for 1h at room temperature (RT) in Licor blocking solution (1:1 with PBST) containing IRDye 800CWDonkey anti-Rabbit or IRDye 680LT Donkey anti-Mouse secondary antibody (1:10000, Licor) as required. Blots were scanned on a Licor Odyssey scanner at a medium resolution setting and exported to ImageJ (NIH).