Molecular dynamics typhoon 9410 molecular imager

After electrophoresis, gels were removed from the cassette and briefly rinsed with water. Fluorescent pictures were taken using the blue laser (488 nm) of a Molecular Dynamics Typhoon 9410 Molecular Imager (GE Healthcare) with a 520 nm bandpass 30 filter for the detection of the fluorescent labeled proteins.

Two hundred μl of Superflow Ni-NTA resin (Qiagen) slurry was added to a 1 ml plastic column. Column was equilibrated with 1 ml native lysis buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0). Cell-free reaction mixture (ERBB2-NLP or YopD-NLP, pre-labeled with FluoroTect^™ GreenLys (Promega)) was added to the prepared Ni-NTA resin and mixed for 1 hour at 4C on a nutator. Columns were then washed with 2 × 200 μl of native lysis buffer with 10 mM imidazole, 2 × 200 μl of native lysis buffer with 20 mM imidazole. Samples were eluted with 2 × 200 μl of either 0.1 M sodium carbonate (pH 11.5), or native elution buffer, lysis buffer with 400 mM imidazole. Eluents were collected and concentrated using Vivaspin 500 spin column (MWCO = 10kD, Sartorius Stedim AG). The concentrated samples resolved by SDA-PAGE. Fluorescent pictures of the gels were taken using the blue laser (488 nm) of a Molecular Dynamics Typhoon 9410 Molecular Imager (GE Healthcare) with a filter 520 nm bandpass 30.