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Rab5 Affinity Chromatography without Nonhydrolyzable GTP Analogues

2009; De Gruyter; Volume: 64; Issue: 3-4 Linguagem: Inglês

10.1515/znc-2009-3-424

1865-7125

Makoto Hagiwara, K. Kobayashi, Tadahiro Tadokoro, Yuji Yamamoto,

Erythrocyte Function and Pathophysiology

Rab5 is an important small GTPase involved in endocytosis and membrane trafficking. Rab5-binding proteins can be identified using Rab5 affinity chromatography with nonhydrolyzable GTP analogues such as GTPγS or GppNHp. However, this method requires significant quantities of the GTP analogue and is thus time-consuming and expensive. In the present report we show a faster and more cost-effective method that does not use a GTP analogue but uses constitutively the active Rab5 mutant (Rab5Q79L) as a ligand. To validate this method, the binding of EEA-1 was confirmed and several novel Rab5-binding proteins were also identified by 2-dimensional electrophoresis and liquid chromatographymass spectrometry/mass spectrometry (LC-MS/MS).