Dictyostelium anti-γ-tubulin Westerns

Harwood lab Protocol, contributed by Emma Dalton, October 2005
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[MATERIALS] - [INDEX]

Procedure

1. Preparation

2. Count 1 x 10⁸ cells, spin down @2000 rpm 2 mins.
3. 
   
4. Talon purify and resuspend in 200 µl. This supernatant is removed and stored at -80°C.
5. 
   
6. Use 10 µl per lane + 10 µl 2x Lamelli. (Visualisation with 5 µl possible but not so strong.)
7. 
   
8. For other samples load approx. 10 µg protein.
9. 
   

   Running and Blotting

10. Take the sample (supernatant and Laemmli mix) and boil for 5 min and cool on ice load each sample onto a 10% acrylamide gel .
11. 
    
12. Run at 100 Volts and when sample/marker is at bottom - stop and transfer to Hybond C+™ membrane.
13. 
    
14. The membrane can then be put into block overnight @ 4°C.
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16. After being in block the membrane should be washed 3 times for 5-10 mins on a rocker each time using 1X TBST.
17. 
    
18. The primary antibody is then added and left 1 hr in a sealed bag @RT.
    
19. 
    
20. After being in the primary antibody, the membrane should be washed again 3 times for 5-10mins on a rocker each time using 1X TBST.
21. 
    
22. The secondary antibody can then be added and left in a sealed bag for 1 hour @RT.
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24. After the secondary antibody, the membrane should be washed again 3 times for 5-10 min on a rocker each time using 1X TBST.
25. 
    

    Visualisation

26. Using Pierce Supersignal kit, the membrane is then placed between pieces of a write-on type overhead transparency and exposures of 5 sec-30 mins can be taken using MR-1 type film.
27. 
    

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Materials

• Blocking solution
  • 1X TBS
  • 0.05% Triton X-100
  • 5% Milk
  NO AZIDE!!!
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• 1X TBST
  • 1X TBS
  • 0.05% Triton X-100
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• Primary antibody
  • Anti-γtubulin (GTU88 from Sigma)@1:1000 in 1X TBST
  Note: If antibody has been warm - change this to 1:500 or it may not work at all!!
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• Secondary antibody
  • anti-Mouse HRP @1:10,000 (Vector labs) in 1X TBST

ECD 08/02

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