Benzamidine

Typically lots of 8–10 bottles, each containing700 ml cultures at ~3.5 – 4x10⁶ cells per ml were harvested by centrifugation at 2500 rpm using a GS3 rotor and washed with cold PBS yielding approximately 80 ml of packed cells. After the PBS wash, cells were resuspended with 10 times the cell pellet volume in ice cold lysis buffer (50 mMTris-HCl pH 7.4, 1 mM EDTA) containing 3X concentrated protease inhibitors: (Leupeptin (RPI) 3.0 μg/ml, Aprotinin (RPI) 6.0 μg/ml, Pefabloc (RPI) 150 μg/ml, Benzamidine (Fisher) 36 μg/ml, E64 (AG Scientific) 10.5 μg/ml. The resuspended cells were incubated on ice for 10 minutes then disrupted with 5 strokes of a 100 ml Dounce homogenizer. Sucrose buffer (25 ml of 1.5 M sucrose, 50 mM Tris pH 7.2) was added to the homogenate and mixed with 2 more strokes. Unbroken cells and cell debris were removed by centrifugation at 5000 rpm in a GS3 rotor. The crude membranes were collected by centrifugation of the GS3 supernatant in a SW28 rotor at 26,500 rpm for 30 minutes at 4°C. The membrane pellets were collected and resuspended in buffer A (see below) containing 2X protease inhibitors). Membranes were stored at −80°C prior to solubilization. Protein concentrations were measured using bicinchoninic acid assay (BCA reagent from Pierce Thermo Scientific).

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE), soy l-α-phosphatidylinositol (PI), 1-oleoyl-2-(6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-2-propionyl)amino)-hexanoyl)-sn-glycero-3-phosphoinositol (TopFluor TMR PI), 1-oleoyl-2-(6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-2-propionyl)amino)-hexanoyl)-sn-glycero-3-phosphatidylethanolamine (TopFluor TMR PE), and 1-palmitoyl-2-{12-[(7-nitro-2–1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphatidylcholine (NBD PC) were purchased from Avanti Polar Lipids (Alabaster, AL). Buffer components tris(hydroxymethyl)-aminomethane (Tris), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium chloride, ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), and benzamidine were purchased from Fisher Scientific (Pittsburgh, PA). Dodecyl thiomaltopyranoside was purchased from Anatrace (Maumee, OH). The buffer solution used for all experiments contained 50 mM Tris, 600 mM HEPES, 300 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 4 mM benzamidine, and 24.7 μM Dodecyl thiomaltopyranoside (pH 8.0) (referred to as Amot ACCH domain elution buffer).