The basic protocol is taken from: Ren X.D., Schwartz M.A. (2000) Determination of GTP loading on Rho. Methods Enzymol. 325, 264-272. 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, 5mM MgCl2, 1% Triton X-100, 0.1% SDS, 10µg/ml aprotinin, 10µg/ml leupeptin, 0.1mM PMSF
TBS: 50mM Tris, pH 7.6, 140mM NaCl
Preparation of GST-RTKN 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 37°C.
- 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 h, check after 45 min).
- Induce with 0.5 mM IPTG (0.5ml of 1M stock) for 2 h.
- 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 s 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 (GE-Healthcare, and pre-equilibrated in bacterial lysis buffer). Rotate for 60 min at 4°C.
- Centrifuge 800 x g, 1 min at 4°C in a refrigerated benchtop centrifuge.
- Wash x6 with 12 ml ice-cold bacterial wash buffer.
- Wash x1 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 800 µl ice-cold Lysis Buffer.
- Centrifuge 15,000 x g, 10 min at 4°C.
- Remove 20 µl from each sample for total Rho protein control.
- Transfer 650 µl lysate into microfuge tube with 20-30 µg GST-RTKN (approximately 200μl bead suspension; 15μl of packed beads).
- Rotate at 4°C for 45 min.
- Wash beads 4 times with 600 µl ice-cold Lysis Buffer.
- Elute by heating at 95°C for 10 min with 50 µl hot SDS-PAGE sample buffer (with 40 mM DTT).
These samples can be used to detect activation of either RhoA, RhoB or RhoC. 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 specificty of the assay comes from the antibody used to detect the Rho. RhoA, B and C are highly homologous and so the antibody must be absolutely specific for the Rho protein in question.
RhoA: Santa Cruz monoclonal anti-RhoA #sc-418
RhoB: Santa Cruz monoclonal anti-RhoB #sc-8048. n.b. Santa Cruz sell two clones of this mAb under the same code. They don’t tell you which you get, but one works much better than the other!
RhoC: currently we do not know of a specific RhoC antibody for these assays.
Troubleshooting and tips
- The hydrolysis of RhoA.GTP is rapid and 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.
- RhoA hydrolysis is aggravated by the high GAP activity of cell lysates. The high salt concentration of the lysis buffer helps to inhibit this (see original Schwartz lab paper).
- You can store the RTKN 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.