Dictyostelium anti-gamma-tubulin Westerns

Dictyostelium anti-γ-tubulin Westerns

Harwood lab Protocol, contributed by Emma Dalton, October 2005
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  1. Preparation

  2. Count 1 x 108 cells, spin down @2000 rpm 2 mins.

  3. Talon purify and resuspend in 200 µl. This supernatant is removed and stored at -80°C.

  4. Use 10 µl per lane + 10 µl 2x Lamelli. (Visualisation with 5 µl possible but not so strong.)

  5. For other samples load approx. 10 µg protein.

  6. Running and Blotting

  7. Take the sample (supernatant and Laemmli mix) and boil for 5 min and cool on ice load each sample onto a 10% acrylamide gel .

  8. Run at 100 Volts and when sample/marker is at bottom - stop and transfer to Hybond C+™ membrane.

  9. The membrane can then be put into block overnight @ 4°C.

  10. After being in block the membrane should be washed 3 times for 5-10 mins on a rocker each time using 1X TBST.

  11. The primary antibody is then added and left 1 hr in a sealed bag @RT.

  12. 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.

  13. The secondary antibody can then be added and left in a sealed bag for 1 hour @RT.

  14. After the secondary antibody, the membrane should be washed again 3 times for 5-10 min on a rocker each time using 1X TBST.

  15. Visualisation

  16. 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.


  • Blocking solution
    • 1X TBS
    • 0.05% Triton X-100
    • 5% Milk
    NO AZIDE!!!

  • 1X TBST
    • 1X TBS
    • 0.05% Triton X-100

  • 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!!

  • Secondary antibody
    • anti-Mouse HRP @1:10,000 (Vector labs) in 1X TBST

ECD 08/02


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