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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
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