Dictyostelium anti-γ-tubulin Westerns

Harwood lab Protocol, contributed by Emma Dalton, October 2005
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Procedure

Preparation

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


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


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


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



Running and Blotting

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


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


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


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


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


10. 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.


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


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



Visualisation

13. 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.

Materials

• 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

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