Smolke:Protocols/Western

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Contents

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

  1. Grow culture (between 5 and 25mL works well) 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
    • Run until dye front is near bottom of gel
  5. 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

  1. Cut out membrane slightly larger than gel
    • Be careful not to touch membrane - use forceps.
  2. Make 2x transfer buffer + 10% MeOH
  3. Pre-equilibrate gel in 2x transfer buffer + 0.02% SDS, 10 minutes
  4. Soak pads + membrane in 2x transfer buffer + 10% MeOH
  5. Layer pad, membrane, gel, pad
  6. Roll a pipet over stack to press out bubbles
  7. Run 15V, 20 minutes
    • Check for transfer - did the (prestained) ladder transfer over?

Blotting

  1. 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)
  2. Wash twice with 1x TBST, 5 minutes (rocking)
  3. Incubate membrane with 10mL 1x TBST + 5% milk + 2μL α-V5 antibody, >1hr (rocking)
    • Can go overnight at 4°C for higher sensitivity
  4. Wash twice with 1x TBST, 5 minutes (rocking)
  5. Mix 2.5mL each developing solution, add to membrane, incubate 5 minutes (rocking)
  6. Take membrane out, place on plastic wrap, blot lightly with Kimwipe. Then fold over plastic wrap to cover (squeeze out bubbles)
  7. 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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