How to measure Rac1 activation state by using a PAK pull-down.
How to measure Rac1 activation state by using a PAK pull-down.
Written by Guillaume Jacquemet
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
- Coat plate with FN (10ug.ml)
- Equilibrate medium with CO2 (3 or 4 time)
- Make fresh buffer buffer supplemented with protease inhibitors (Apoprotinin 10ug/ml; leupetine 10ug/ml; AEPBSF 0.5mM)
- Pre-label tubes, for each condition prepare one tube for:
- The cell lysis
- The total extract
- The pull-down
- The 1st supernatant
- Add 30ul of PAK beads and 100ul of Rac1 buffer into the pull-down tubes
- Keep all tubes on ice
Prepare cells
- Wash cells with PBS-
- Trypsin cells
- Pellet cells at 1800rpm
- Resuspend cells in a small volume of media and count them
- Keep cells on suspension at 37°C for about 30min
- Plate cells at the same density for all the conditions on a 100cm plate
PAK pull-down
- Wash cells with cold PBS (aspirate as much as possible)
- Add 140ul of cold lysis buffer for each plate
- Scrape plate on ice, and put the cell lysate into the “cell lysis tube”
- Clear the lysate by centrifugation (14,000rpm, 4°C)
- Transfer the supernatant into the “total extract tube”
- Transfer 120uL of the lysate into the “pull-down tube” (which contained the PAK beads)
- Rotate at 4°C for 1H
- Pellets the beads at 7000rpm 4°C for 1min
- Wash the beads 3 times with 500uL of cold Arf6 washing buffer
- Elute GTP bound PAK with 50uL of 2X SDS loading buffer
- Boil sample by using water bath for 5min
- Store samples at 4°C prior to analysis
Analysis
- Run, for each condition, equal volumes of the GTP-Rac1 eluted from PAK beads on a SDS-PAGE gel
- Run, for each condition, equal volumes of the “total extract” on a SDS-PAGE gel
- Realise a western blot with an anti- Rac1 antibody (BD Biosciences 610651)
- Calculate the ratio between GTP- Rac1 and total Rac1 to estimate the Rac1 activation state
How to measure Rac1 activation state by using a PAK pull-down – right hand column
