Smolke:Protocols/Western

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Still a work in progress Overview Blotting for large 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 culture (between 5 and 25mL works well) in appropriate (generally dropout) media Pellet cells at 3000g, 4°C for 5 minutes Pour off supernatant, weigh, resuspend in water at 1 mg/uL 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 Run until dye front is near bottom of gel Crack open gel, trim off top (wells) and bottom of gel with razor Can trim gel down further if all lanes weren't used Semi-dry Transfer Cut out membrane slightly larger than gel Be careful not to touch membrane - use forceps. 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 Roll a pipet over stack to press out bubbles Run 15V, 20 minutes Check for transfer - did the (prestained) ladder transfer over? Blotting Incubate membrane with 1x TBST + 5% milk, >1hr (rocking) Make sure there are no clumps in the milk (particularly a problem if you store the TBST+milk) Wash twice with 1x TBST, 5 minutes (rocking) Incubate membrane with 10mL 1x TBST + 5% milk + 2μL α-V5 antibody, >1hr (rocking) Can go overnight at 4°C for higher sensitivity Wash twice with 1x TBST, 5 minutes (rocking) Mix 2.5mL each developing solution, add to membrane, incubate 5 minutes (rocking) Take membrane out, place on plastic wrap, blot lightly with Kimwipe. Then fold over plastic wrap to cover (squeeze out bubbles) Image membrane Use gel imager on chemiluminescence setting. Set for 5-100 s, in 5s increments It's also useful to take another picture in white light (without moving membrane) to image the ladder. Notes References Contact Josh Michener