Odom:Western Blot

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Revision as of 18:02, 12 September 2006

Contents

Overview

This is the western blot protocol used in the Odom Lab. Standard protocol for use with pre-cast gels.

Materials

Sample Buffer

  • 950ul Laemmli Sample Buffer (Biorad)
  • 50ul BME

Running Buffer

  • 150ml 10X Tris/Glycine/SDS
  • 1350ml water

Transfer Buffer (10X mix, 20% methanol)

  • 200ml 10X Tris/Glycine
  • 400ml methanol
  • 1400ml water

Blocking Buffer (5% milk in TBS)

  • 2.5g nonfat dry milk (5%)
  • up to 50ml TBS

Ponceau S Solution

  • Dissolve 0.5g Ponceau S in 1ml glacial acetic acid
  • Bring to 100ml with water

TBST

  • 500ul Tween-20
  • 1L TBS

Primary Wash (0.5% milk, 0.01% NaAz in TBST)

  • 0.5g nonfat dry milk
  • up to 10ml in TBST
  • 20ul 5% sodium azide
  • primary antibody

Secondary Wash (5% milk in TBST)

  • 1g nonfat milk (5%)
  • up to 20ml with TBST
  • secondary antibodies

Procedure

  1. Add Sample Buffer to lysate and boil at 95 for 5min.
    • ~30-100ug protein/lane
    • 2 volumes sample buffer
    • PBS can be added to add volume to lysate
  2. Add Running Buffer and load gel
  3. Run at 60-100v for ~3 hours in the cold room
  4. Make transfer buffer and chill to 4
  5. Wet nitrocellulose membrane in ddH20 then transfer buffer for 1-15min
  6. Disassemble gel by pushing opening down onto lid of transfer apparatus
  7. Cut the stacking gel off
  8. Assemble transfer apparatus
    • Sponge
    • Whatman
    • Gel
    • Nitrocellulose membrane, pre-wet
    • Whatman
    • Sponge
    • Remove all air bubbles with each new layer
    • Membrane must be on positive side, gel on negative side
  9. Fill tank with Transfer buffer
  10. Transfer for 3.5 hours at 300mA or O/N at 100mA
  11. Disassemble apparatus and wet membrane in TBS for 5min
  12. OPTIONAL: Visualize transferred proteins with Ponceau stain
    1. Place membrane in Ponceau S solution for 5min at RT
    2. Destain in water for 2min
    3. Completely destain in water for additional 10mins
  13. Block with blocking buffer for 2 hours at RT or O/N in cold
  14. Wash with TBST for 15min RT
  15. Block with Primary Wash for 3 hours at RT or O/N in cold
  16. Wash in TBST 4x15min at RT
  17. Wash with Secondary Wash for 1 hour at RT
  18. Wash in TBST 4x15min at RT
  19. Combine luminal solutions solutions 1:1 to make 5ml and drip over the membrane
  20. Cover with saran wrap and let sit in dark for 3min
  21. Dab off excess luminal and visualize in darkroom immediately
    • 2min is good starting exposure

Notes

  • Please add user notes and tips.

References

  • Also see:

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