Martin Humphries
LAB

Protocol - How to mesure Rac1 activation state by using a PAK pull-down.

Written by Guillaume Jacquemet


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Rac1 activity during cell spreading on Fibronectin


- Buffers:


Rac1 Buffer
  • 20mM Hepes pH=7.5
  • 140mM NaCl
  • 1% NP40 (Igepal CA-630)
  • 4mM EDTA
  • 4mM EGTA
  • 0.5% Deoxycholate
  • 10% glycerol

- Set up the experiment


  1. Coat plate with FN (10ug.ml)
  2. Equilibrate medium with CO2 (3 or 4 time)
  3. Make fresh buffer buffer supplemented with protease inhibitors (Apoprotinin 10ug/ml; leupetine 10ug/ml; AEPBSF 0.5mM)
  4. Pre-label tubes, for each condition prepare one tube for:
    • The cell lysis
    • The total extract
    • The pull-down
    • The 1st supernatant
  5. Add 30ul of PAK beads and 100ul of Rac1 buffer into the pull-down tubes
  6. Keep all tubes on ice


- Prepare cells


  1. Wash cells with PBS-
  2. Trypsin cells
  3. Pellet cells at 1800rpm
  4. Resuspend cells in a small volume of media and count them
  5. Keep cells on suspension at 37°C for about 30min
  6. Plate cells at the same density for all the conditions on a 100cm plate


- PAK pull-down


  1. Wash cells with cold PBS (aspirate as much as possible)
  2. Add 140ul of cold lysis buffer for each plate
  3. Scrape plate on ice, and put the cell lysate into the "cell lysis tube"
  4. Clear the lysate by centrifugation (14,000rpm, 4°C)
  5. Transfer the supernatant into the "total extract tube"
  6. Transfer 120uL of the lysate into the "pull-down tube" (which contained the PAK beads)
  7. Rotate at 4°C for 1H
  8. Pellets the beads at 7000rpm 4°C for 1min
  9. Wash the beads 3 times with 500uL of cold Arf6 washing buffer
  10. Elute GTP bound PAK with 50uL of 2X SDS loading buffer
  11. Boil sample by using water bath for 5min
  12. Store samples at 4°C prior to analysis


- Analysis


  1. Run, for each condition, equal volumes of the GTP-Rac1 eluted from PAK beads on a SDS-PAGE gel
  2. Run, for each condition, equal volumes of the "total extract" on a SDS-PAGE gel
  3. Realise a western blot with an anti- Rac1 antibody (BD Biosciences 610651)
  4. Calculate the ratio between GTP- Rac1 and total Rac1 to estimate the Rac1 activation state



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