Smolke:Protocols/Western

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

Revision as of 18:23, 23 January 2009

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Still a work in progress

Contents

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

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