The basic protocol is taken from: Benard V and Bokoch GM (2002) Assay of Cdc42, Rac, and Rho GTPase activation by affinity methods. Methods Enzymol. 345, 349-359. PMID.
Bacterial Lysis Buffer: 50 mM Tris, pH 7.5, 150mM NaCl 1% Triton X-100, 5mM MgCl2, 1mM DTT, 10μg/ml aprotinin, 10μg/ml leupeptin, 0.1mM PMSF
Bacterial Wash Buffer: 50 mM Tris, pH 7.5, 150mM NaCl 0.5% Triton X-100, 5mM MgCl2, 1mM DTT, 10μg/ml aprotinin, 10μg/ml leupeptin, 0.1mM PMSF
Lysis Buffer: 50mM Tris, pH 7.2, 500mM NaCl, 10mM MgCl2, 1% Triton X-100, 10μg/ml aprotinin, 10μg/ml leupeptin, 0.1mM PMSF
TBS: 50mM Tris, pH 7.6, 140mM NaCl
Tris buffer: 50mM Tris, pH 7.2, 150mM NaCl, 10mM MgCl2, 1% Triton X-100, 10μg/ml aprotinin, 10μg/ml leupeptin, 0.1mM PMSF.
Preparation of GST-PAKCRIB beads
- Inoculate from a single colony of E. coli (BL21(DE3)pLysS) containing the GST-RTKN construct into 5 ml LB broth containing 100μg/ml ampicillin and 34μg/ml chloramphenicol.
- Incubate with shaking overnight at 37°C.
- Inoculate 400 ml LB broth containing 100μg/ml ampicillin and 34μg/ml chloramphenicol.
- Incubate with shaking overnight at 37°C. Prewarm 4 litres LB overnight at 37oC.
- Dilute overnight culture 1:10 into 3600 ml LB containing 100μg/ml ampicillin in 4 x 2 litre flasks.
- Grow to OD 600=0.8 (approx 1 hr, check after 45 min).
- Induce with 0.5 mM IPTG (0.5ml of 1M stock) for 2 hrs.
- Harvest by centrifugation 2,500 x g, 25 min.
- Resuspend completely in 40 ml ice-cold bacterial lysis buffer.
- Transfer to 2 prechilled 40ml Oakridge centrifuge tubes.
- Sonicate on ice 6-8 times for 15 sec each (setting 3 on our machine). Cool 1-2 min between sonications.
- Centrifuge 17,000 x g for 30 min at 4°C.
- Mix lysate with 0.6 ml (packed bead volume) glutathione-Sepharose 4B beads (Amersham, and pre-equilibrated in bacterial lysis buffer). Rotate for 60 min at 4°C.
- Centrifuge 800 x g, 1 min at 4 oC in Beckman refrigerated benchtop centrifuge.
- Wash x 6 with 12 ml ice-cold bacterial wash buffer.
- Wash x 1 with 12 ml bacterial wash buffer + 10% glycerol.
- Resuspend in 8 ml bacterial wash buffer + 10% glycerol.
- Aliquot into prechilled tubes and store at -70°C.
Preparation of Cell Lysates
An ideal starting point is 1 x 10cm dish of cells per condition (approximately 10 million cells), although you should be able to get away with a quarter of this.
- Wash cells twice in ice-cold TBS. Drain.
- Extract in 800μl ice-cold Lysis Buffer.
- Centrifuge 15,000 x g, 10 min at 4°C.
- Remove 20μl from each sample for total Rho protein control.
- Transfer 650μl lysate into microfuge tube with 20-30μg GST-PAKCRIB beads (approximately 200μl packed bead volume).
- Rotate at 4°C for 45 min.
- Wash beads 4 times with 600μl ice-cold Tris Buffer.
- Elute by heating at 95°C for 10 min with 50μl hot SDS-PAGE sample buffer (with 40 mM DTT).
These samples can be used to detect activation of either Rac or Cdc42. Samples are resolved by SDS-PAGE (we use 12% Novex precast Bis-Tris gels) and transfered to PVDF membranes (Immobilon-P) for western blotting. The specificity of the assay comes from the antibody used to detect the Rho. The antibody must be absolutely specific for the Rho protein in question.
Rac1: Becton Dickinson monoclonal anti-Rac1 #R56220
Cdc42: Becton Dickinson monoclonal anti-Cdc42 #610928
G25K: (splice variant of Cdc42) Santa Cruz Cdc42 (c-20) goat polyclonal antibody #sc-6083
Troubleshooting and tips
- The hydrolysis of these Rho GTPases is rapid so it is critical to perform these assays as quickly as possible. Don’t attempt to many samples, and process the lysates you have immediately. You can buy empty microfuge spin columns (e.g. BioRad #732-6204) which allow you to wash the samples quickly and effectively without losing the beads.
- You can store the PAK CRIB beads in liquid nitrogen for months. Surprisingly, it works better to freeze the protein on the beads.
- If you are having trouble seeing Rho GTPases on your western blot, try adding 10% methanol to your transfer buffer.
- Rac is the easiest Rho GTPase to assay and it many systems it undergoes big changes in activity. Cdc42 activations tend to be smaller fold-changes – maybe affecting only a fraction of the cellular pool. This makes them much harder to detect. An alternative for Cdc42 is to use WASP CRIB domain beads.
- It is important to remember that Cdc42 is present as two splice forms in cells, which vary at their C-terminus regions. Some commercial antibodies are (unintentionally) specific for one form; some recognise both.