Complete edta free protease inhibitor

Dissected mouse brain regions were homogenized by performing 12 strokes with a Dounce homogenizer containing 2 mL ice-cold homogenization buffer (320 mM sucrose, 1 mM HEPES, pH 7.4) containing 1× Complete EDTA-free protease inhibitor (Roche) and 1× Phosphatase inhibitor cocktail set II (Calbiochem). Synaptosomes were isolated from homogenized mouse brain tissue as described [2 (link)]. Briefly, insoluble material was pelleted by centrifugation at 1000× g for 10 min at 4 °C. The supernatant (S1) was removed and the pellet resuspended in 1 mL homogenization buffer and an additional six strokes were performed. Following a second centrifugation at 1000× g for 10 min at 4 °C, the supernatant (S2) was removed and pooled with S1. The combined supernatants were then centrifuged at 18,500× g for 15 min at 4 °C. The pellet was resuspended in 0.25 mL homogenization buffer and 0.25 mL extraction buffer (50 mM NaCl, 1% DOC, 25 mM Tris-HCl, pH 8.0) containing 1× Complete EDTA-free protease inhibitor (Roche) and 1× Phosphatase inhibitor cocktail set II (Calbiochem) and incubated on ice for 1 h. The resulting PSD extracts were centrifuged at 10,000× g for 20 min at 4 °C and the resulting supernatant filtered through a 0.2 µm syringe filter (Millipore).

For nuclear extract preparation, MEL cells were harvested by centrifugation at 1000g and washed once in 1× phosphate-buffered saline. Cell pellets were resuspended in chilled resuspension buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, Complete EDTA free protease inhibitor (Roche), 1 mM dithiotreitol (DTT)). Cell suspension was incubated on ice for 10 min, mixed occasionally and vortexed for 10 s. The nuclei were pelleted by centrifugation for 1 min at 16 300g and resuspended in 20 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 420 mM NaCl, 25% glycerol, Complete EDTA free protease inhibitor (Roche), 1 mM DTT and incubated for 1 h at 4°C on a rotary shaker. Nuclear extracts were obtained by centrifugation at 16 300g for 5 min and supernatants aliquoted and stored at −80°C.

Crude PSD preparations were made from dissected mouse forebrain tissue from C57BL6/5J mice ages 1 month to 5 months. In brief, each forebrain was homogenized by performing 12 strokes with a Dounce homogenizer containing 5 mL of ice cold homogenization buffer (320 mM sucrose, 1 mM HEPES, pH = 7.4) containing 1X Complete EDTA-free protease inhibitor (Roche) and 1X Phosphatase inhibitor cocktail set II (Calbiochem). Insoluble material was pelleted by centrifugation at 1000 x g for 10 min at 4˚C. The supernatant (S1) was removed and the pellet was re-suspended in 2 mL of homogenization buffer and an additional six strokes were performed. Following a second centrifugation at 1000 x g for 10 min at 4˚C, the supernatant (S2) was removed and pooled with S1. The combined supernatants were then centrifuged at 18, 500 x g for 15 min at 4˚C. The pellet was re-suspended in 5 mL of extraction buffer (50 mM NaCl, 1% DOC, 25 mM Tris-HCl, pH 8.0) containing 1X Complete EDTA-free protease inhibitor (Roche) and 1X Phosphatase inhibitor cocktail set II (Calbiochem) and incubated on ice for 1 hr. The resulting crude PSD extracts were centrifuged at 10,000 x g for 20 min at 4 ˚C and the resulting supernatant filtered through a 0.2 µm syringe filter (Millipore).

BL21 (DE3) E. coli cells were co-transformed with both pET17-twin-strep-C16 and pET28b-flag-C4 and grown in standard LB medium supplemented with ampicillin and kanamycin. Protein expression was induced upon the addition of IPTG at 0.5 mM final concentration once cells had reached an optical density (600 nm) of 0.8–1.0 and the temperature had been reduced from 37 to 16 °C. After growth overnight, cells were pelleted by centrifugation and then resuspended in buffer A (50 mM HEPES-KOH pH 7.5, 500 mM NaCl, 5% (w/v) glycerol, 0.5 mM TCEP), supplemented with lysozyme, benzonase nuclease (Merck Life Sciences) and cOmplete^TM EDTA-free protease inhibitor (Roche). Cells were lysed by sonication and cleared by centrifugation.
Pull-downs were performed using the cell lysate from the C16 and C4 co-expression. The lysate was divided in half, and then applied to either Strep-Tactin beads (IBA, catalog number 2-1613-002, 1 ml to obtain a bed volume of 50 µl) or Anti-Flag beads (Merck, catalog number M8823, 100 µl to obtain a bed volume of 50 µl). After extensive washing, proteins retained on the beads were eluted with the addition of either biotin or flag peptide respectively and the eluted proteins analysed by SDS-PAGE and immunoblotting with either anti-strep (Merck, catalog number SAB27002216, 1:7000 dilution) or anti-flag antibodies (Merck, catalog number F3165, 1:5000 dilution).