Rab5 affinity chromatography without nonhydrolyzable GTP analogues.
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 GTPgammaS 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 chromatography-mass spectrometry/mass spectrometry (LC-MS/MS).
Hagiwara
Makoto
M
Department of Agricultural Chemistry, Graduate School of Agriculture, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan.
Kobayashi
Ken-Ichi
K
Tadokoro
Tadahiro
T
Yamamoto
Yuji
Y
D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D003600:Cytosol / Q000502:physiology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives; D006165:Guanylyl Imidodiphosphate; D013154:Spleen / Q000502:physiology; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification*
D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D020696:rab5 GTP-Binding Proteins