Glucosylation of Recombinant GTPases for Microinjection

Recombinant Rho proteins are glucosylated in the presence of unlabeled UDP-glucose. The reaction is terminated by passing the reaction mixture through a membrane with an exclusion molecular weight of 100 kDa to remove toxin B (270 kDa). As a control, the GTPase is incubated with toxin B but in absence of the cosubstrate UDP-glucose.

Recombinant Rho proteins (400|xg/ml) are glucosylated in a buffer containing 2mM MgCI2, 0.1 mM GDP, 50 mM HEPES (pH 7.4), 0.1 mM UDP-glucose and 5|^g/ml toxin B (total volume lOÖjxl) for 45 min at 37°C. Thereafter, the reaction mixture is centrifuged at 5000g in a Microcon 100 (Amicon). The filtrate (<100kDa) contains the GTPase but not toxin B. The sample is now ready for microinjection or can be concentrated further prior to microinjection using Microcon 10 (Amicon).

The effects of microinjected glucosylated Rho GTPases are monitored by detection of morphological changes (see section 13.4) or by visualization of the microfilament system with rhodamine- or FITC-phalloidin (for staining see Chapter 10).

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