Smolke:Protocols/Western

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Still a work in progress Overview Blotting for V5-tagged proteins in S. cerevisiae Materials Y-PER (Pierce) Halt EDTA-free Protease Inhibitor (Pierce) NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX) Prestained protein ladder (Invitrogen LC5800) Protein loading buffer MOPS buffer (Invitrogen NP0001) Nitrocellulose membrane Blotting pads Transfer buffer (Invitrogen NP0006-1) Methanol Anti-V5-HRP antibody (Invitrogen R961-25) Chemiluminescence detection kit (Pierce) Procedure Lysis Grow 25mL culture to saturation in appropriate (generally dropout) media. Pellet cells at 3000g, 4°C for 5 minutes Pour off supernatant, weigh, resuspend in water at 1 mg/mL. Transfer 100uL to a 1.5mL tube, repellet as before Reweigh, resuspend at 0.5 mg/uL in YPER (+EDTA-free protease inhibitor) Agitate at RT for 20min Pellet at 14000g, 4°C for 10 minutes SDS-PAGE Remove 40uL of lysate supernatant, mix with 10uL of 5x loading buffer Also take 5uL prestained ladder (thaw and vortex first) Boil samples while prepping precast SDS-PAGE gel Load 25uL of each sample Run gel with MOPS running buffer, 150V, ~1hr Semi-dry Transfer Make 2x transfer buffer + 10% MeOH Pre-equilibrate gel in 2x transfer buffer + 0.02% SDS, 10 minutes Soak pads + membrane in 2x transfer buffer + 10% MeOH Layer pad, membrane, gel, pad Run 15V, 20 minutes Blotting Incubate membrane with 1x TBST + 5% milk, >1hr (rocking) Wash twice with 1x TBST, 5 minutes (rocking) Incubate membrane with 10mL 1x TBST + 5% milk + 2μL α-V5 antibody, >1hr (rocking) Wash twice with 1x TBST, 5 minutes (rocking) Mix 2.5mL each developing solution, add to membrane, incubate 5 minutes (rocking) Image Notes References Contact Josh Michener