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Millipore Technical Publications

Protocol: General Method for Ras, Rac, Cdc42, and Rho Activation Assays
Catalogue Number: mcproto416

Affinity Precipitation/Immunoblot Protocol
A. Stock Solutions
  1. 5X Mg2+ Lysis/Wash Buffer (5X MLB, catalog # 20-168): Dilute to 1X by adding 4ml Milli-Q water (or equivalent) containing 10% glycerol to each ml of 5X Mg2+ Lysis/Wash Buffer. To the diluted buffer (MLB) add 10 µg/ml aprotinin and 10 µg/ml leupeptin. Optionally, the phosphatase inhibitors 25mM sodium fluoride and 1mM sodium orthovanadate may also be added to the buffer.
  2. 100XGTPyS ,10mM(catalog#20-176): Use 5 µl of GTPyS for GTP-labeling 0.5ml cell lysate.
  3. 100X GDP, 100mM(catalog #20-177):Use 5 µl of GDP for GDP-labeling 0.5ml cell lysate.
  4. 1 M MgCl 2: Formulate using deionized water.
  5. 0.5 M EDTA, pH 8.0: Formulate using deionized water.
Cell Culture and Extract Preparation
Note: While Upstate recommends using fresh lysates because GTP is rapidly hydrolyzed to GDP, frozen lysates may be used, provided that the lysates were snap frozen in liquid nitrogen and stored at or below -70°C. Performing all steps at 4 ° C or on ice may reduce hydrolysis. To bind the GTP-bound form of either Ras, Rac, cdc42, and/or Rho, add the individual specific protein binding domain/agarose directly to the lysate immediately after removing cellular debris and insoluble material by centrifugation. A sample of the lysates may be saved for assaying protein concentration, and protein loading may be equalized at the time of loading the gel.
Adherent Cells
  1. Culture cells to approximately 85-90% confluence, stimulating and GTPase activation as desired.
  2. Remove culture media, rinse twice with ice-cold Tris-Buffered Saline (TBS).
  3. Add ice-cold MLB (0.5-1ml per 150mm tissue culture plate) to rinsed cells in plates on ice.
  4. Detach (and lyse) the cells from plates by scraping with a rubber policeman or cell scraper.
  5. Transfer the lysates to microfuge tubes on ice. Proceed to Step 6.
Non-Adherent Cells
  1. Culture cells and stimulate GTPase activation as desired.
  2. Pellet cells by gentle centrifugation (500xg), then rinse twice with ice-cold Tris-Buffered Saline (TBS).
  3. Discard supernatant, and add ice-cold MLB to the cell pellet (0.5-1ml per 10 7 cells).
  4. Lyse cells by repeated (4-5 times) pipetting.
  5. Transfer the lysates to microfuge tubes on ice. Proceed to Step 6.
    NOTE: If nuclear lysis occurs, the extract may be very viscous (and difficult to pipette) due to released genomic DNA. DNA may be sheared by passing the lysate through a 26-gauge syringe needle 3-4 times (prior passage through larger-bore needles may be required first).
  6. Clear lysates of insoluble cell debris by centrifugation (5 minutes, 14,000xg, 4 °C).
  7. Remove the extract (supernatant), and store aliquots on ice (for immediate use) or snap freeze in liquid nitrogen. Frozen extracts may be stored at –70 °C or lower (for long term).
GTPyS/GDP Loading for Positive and Negative Controls
Note: Samples that will not be loaded with GTPyS/GDP may be kept on ice during the loading of controls.
  1. Aliquot 0.5ml of each cell extract to two microfuge tubes.
  2. To each tube, add 10 µl of 0.5M EDTA (to 10mM, final concentration).
  3. Add 5 µl of 100X GTPyS (to 100 µM, final concentration) to one tube (positive control).
  4. Add 5 µl of 100X GDP (to 1mM, final concentration) to a second tube (negative control).
  5. Incubate the tubes for 30 minutes at 30 oC with agitation.
  6. Stop loading by placing the tubes on ice and adding 32 µl of 1M MgC l 2 (to 60mM, final concentration).
Ras, Rac ,Cdc42, and Rho Pull-Down Assay
Note: The optimal conditions for a specific cell system and GTPases have to be determined empirically. The active form of the GTPase is labile, and there is rapid turnover of the GTP-bound state, which makes its measurement difficult.
  1. Aliquot 0.5ml of each cell extract to a microfuge tube.
  2. Add 30-50 µl (20-30 µg) of the GTPase Protein Binding Domain/agarose slurry of interest.
  3. Incubate the reaction mixtures for 45 minutes at 4°C with gentle agitation.
  4. Pellet the agarose beads by brief centrifugation (10 seconds, 14,000xg, 4°C).
  5. Remove and discard the supernatant.
  6. Wash the beads (add 0.5ml MLB, mix gently, pellet beads, remove MLB) 3 times with MLB. Take care to minimize loss of beads when MLB is removed.
  7. Resuspend the agarose beads in 40 µl of 2X Laemmli reducing sample buffer and boil for 5 minutes. Pellet the beads by brief centrifugation. Addition of 2 µl of 1M dithiothreitol prior to boiling may improve release of Rho from the beads.
  8. Centrifuge briefly to collect condensate in the bottom of the tube.
Western Blot and Detection
  1. Mix the supernatant and the agarose pellet and load 20 µl of the mixture per lane on an appropriate polyacrylamide gel.
    NOTE: Adding beads to gel will not effect electrophoresis. Sometimes the affinity precipitated GTPase may re-associate with the protein binding domain on the agarose after boiling. Loading slurry will help maintain consistent results.
  2. Perform SDS-PAGE and western blot to a nitrocellulose (or other) membrane.
  3. Rinse the membrane twice with water.
  4. Incubate the membrane 30 minutes at room temperature with agitation in freshly prepared 3% nonfat dry milk (Upstate, Cat. No. 20-200) in PBS (PBS-MLK).
  5. Incubate the membrane overnight at 4 °C with agitation with either the anti-Ras, Rac, Cdc42, and/or Rho diluted in freshly prepared PBS-MLK.
  6. Wash the membrane twice (5-10 minutes each) with PBS.
  7. Incubate the membrane for 1 hour at room temperature with agitation in the appropriate secondary antibody conjugate (a goat anti-rabbit-HRP conjugate is recommended, Cat. No. 12-348. The optimal dilution must be empirically determined by titration/dilution to find the best optimal dilution of secondary antibody in PBS-MLK.
  8. Wash the membrane 3 times (5-10 minutes each) with PBS.
  9. Use a detection method of choice. HRP conjugates are indicated for the secondary antibody, so an appropriate luminol-based chemiluminescent HRP substrate solution is recommended (colorimetric detection may be used, but sensitivity will be reduced, as compared with a chemiluminescent detection).