Odom:Western Blot

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:

Contact

• cmc