Martin Humphries
LAB

Protocols - Arf6 effector pull-down assays

Written by Guillaume Jacquemet


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Arf6 activity during cell spreading on Fibronectin


- Buffers:


Arf6 Lysis Buffer
  • 50mM Tris pH=7.5
  • 150mM NaCl
  • 10mM MgCL2
  • 1% triton
  • 0.5% Deoxycholate
  • 0.1% SDS
  • 10% glycerol
Arf6 Washing buffer
  • 50mM Tris pH=7.5
  • 100mM NaCl
  • 2mM MgCL2
  • 1% NP40 (Igepal CA-630)
  • 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 buffers 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 pulldown
    • The 1st supernatant
  5. Add 30ul of GGA3 beads and 100ul of Arf6 lysis buffer into the pulldown 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


- GGA3 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 GGA3 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 Arf6 with 50uL of 2X SDS loading buffer
  11. Boil samples by using water bath for 5min
  12. Store samples at 4°C prior to analysis


- Analysis


  1. Run, for each condition, equal volume of the GTP-Arf6 eluted from GGA3 beads on a SDS-PAGE
  2. Run, for each condition, equal volume of the "total extract" on a SDS-PAGE
  3. Realise a western blot with an anti- Arf6 antibody (ARFAG sigma A5230)
  4. Calculate the ratio between GTP-Arf6 and total Arf6 to estimate the Arf6 activation state


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