Gfp polyclonal antibody

The 501mel human melanoma cells expressing doxycycline-inducible human GFP-tagged MITF or TFEB were treated with 0.2 μl.ml⁻¹ doxycycline and transfected with mouse FLAG-tagged MITF(wt), MITF(Δ), or human FLAG-tagged MAX 24 h after seeding. The 501 human melanoma cell line was co-transfected with mouse GFP-tagged MITF(Δ) and mouse FLAG-tagged MITF(wt) or human FLAG-tagged MAX. Cells were washed 48 h after transfection with ice-cold PBS and lysed on a shaker for 15 min with coIP lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton) supplemented with PMSF and a protease inhibitor cocktail. The lysates were scraped and centrifuged at 14 000×g for 10 min. A fraction of each supernatant was collected and stored (input). The remaining lysates were incubated at 4°C on a rotating platform with 2.5 μg GFP polyclonal antibody (Abcam) for 3 h. Subsequently, 20 μl of A/G plus-agarose beads (Santa-Cruz Biotechnology) were added and samples incubated for 1 h at 4°C on a rotating platform. Samples were centrifuged (2500 rpm for 5 min) to collect the beads and then washed three times with TBS buffer. Samples were eluted with 25 μl of 2× Laemmli buffer, boiled for 5 min and run on a gel for immunoblotting with GFP, FLAG and actin antibodies.