Smolke:Protocols/Western

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

1. Grow culture (between 5 and 25mL works well) to saturation in appropriate (generally dropout) media
2. Pellet cells at 3000g, 4°C for 5 minutes
3. Pour off supernatant, weigh, resuspend in water at 1 mg/uL
4. Transfer 100uL to a 1.5mL tube, repellet as before
5. Reweigh, resuspend at 0.5 mg/uL in YPER (+EDTA-free protease inhibitor)
6. Agitate at RT for 20min
7. Pellet at 14000g, 4°C for 10 minutes

SDS-PAGE

1. Remove 40uL of lysate supernatant, mix with 10uL of 5x loading buffer
   • Also take 5uL prestained ladder (thaw and vortex first)
2. Boil samples while prepping precast SDS-PAGE gel
3. Load 25uL of each sample
4. Run gel with MOPS running buffer, 150V, ~1hr

Semi-dry Transfer

1. Make 2x transfer buffer + 10% MeOH
2. Pre-equilibrate gel in 2x transfer buffer + 0.02% SDS, 10 minutes
3. Soak pads + membrane in 2x transfer buffer + 10% MeOH
4. Layer pad, membrane, gel, pad
5. Run 15V, 20 minutes

Blotting

1. Incubate membrane with 1x TBST + 5% milk, >1hr (rocking)
2. Wash twice with 1x TBST, 5 minutes (rocking)
3. Incubate membrane with 10mL 1x TBST + 5% milk + 2μL α-V5 antibody, >1hr (rocking)
4. Wash twice with 1x TBST, 5 minutes (rocking)
5. Mix 2.5mL each developing solution, add to membrane, incubate 5 minutes (rocking)
6. Image

Notes

References

Contact

Josh Michener