Egfp antibody

Liposome generation and flotation was performed as described in Vollmer et al., 2015 (link). In short, E. coli polar lipids (Avanti Polar Lipids) dissolved in chloroform and supplemented with 0.2 mol% 18:1 Liss Rhodamine PE (Avanti Polar Lipids) were vacuum-dried on a rotary evaporator, dissolved as liposomes in PBS by freeze/thawing cycles and extruded by passages through Nuclepore Track-Etched Membranes (Whatman) with defined pore sizes using an Avanti Mini-Extruder to generate small unilamellar liposomes of defined sizes. For liposome flotations, proteins (6 μM) were mixed 1:1 with liposomes (6 mg/mL) and floated for 2 hr at 55,000 rpm in a TLS-55 rotor (Beckman) at 25°C through a sucrose gradient. Binding efficiency was determined by Western blot analysis using an EGFP antibody (Roche, 11814460001, 1:2000). As secondary antibody, an anti-mouse, horseradish peroxidase conjugated antibody (Calbiochem, 401215, 1:5000) was used. The ImageQuant LAS-4000 system (Fuji) and the AIDA software were used to compare band intensities of start materials with floated liposome fraction.

Cells transfected with plasmid DNA were lysed in RIPA buffer with the addition of protease inhibitor and phosphatase inhibitor (Roche). The cell extracts were harvested by centrifugation at 4°C for 10 min and the supernatant was used for IP assay. Cell extracts (500 μg) were incubated with 20 μl of anti-Flag M2 affinity gel (50% slurry) (Sigma-Aldrich, St. Louis, MO) at 4°C for 2 h. Immunoprecipitates were collected by centrifugation at 1000 × g, washed with RIPA buffer for three times followed by SDS-PAGE electrophoresis and western blot. For co-IP assay, cells were co-transfected with equal amount of Tag-fusioned constructs and lysed 48 h later. anti-Flag M2 affinity gel (50% slurry) (Sigma-Aldrich) and EGFP antibody (Roche) were used for IP of protein complex from cell extract at 4°C for 2 h. After washing with PBS for three times, the immunoprecipitates were eluted by sampling buffer and subject to SDS-PAGE and western blot.