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- W58602382 abstract "This chapter discusses the expression and properties of Rab4 and its effector Rabaptin-4 in endocytic recycling. To investigate binding of Rab4 to the N terminus of Rabaptin-4 in vitro, the expression plasmid pRSET/ C-Rabaptin-4AC is generated. This construct encodes amino acids 11–388 of Rabaptin-4 and is prepared by releasing an EcoRI-HindIII fragment and ligating it in the same sites of pRSET/C. For the expression of His 6 -tagged Rab4 and His 6 -tagged Rabaptin-4ΔC, starting with fresh transformants is recommended because they provide the most reproducible results in terms of protein yield. To increase the yield of His 6 -tagged Rabaptin-4ΔC cultures are grown at room temperature; however, this provides little improvement. For purification of His 6 -tagged Rab4, all buffers are supplemented with 5 μM GDP to stabilize the small GTPase. Typical yields are 1.5 mg of His 6 - tagged Rab4 and 0.3 mg of His 6 -tagged Rabaptin-4ΔC per liter of culture. In addition, a simple and rapid procedure to assay guanine nucleotide-dependent binding of Rab4 to Rabaptin is developed using an enzyme-linked immunosorbent assay (ELISA) format in which His 6 -tagged Rabaptin-4 is coated in microtiter plates. Subsequently wells are incubated with increasing amounts of His 6 -tagged Rab4 in the presence of GDP or GTP and bound Rab4 is determined with Rab4 antibody." @default.
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- W58602382 date "2001-01-01" @default.
- W58602382 modified "2023-09-27" @default.
- W58602382 title "[13] Expression and properties of Rab4 and its effector rabaptin-4 in endocytic recycling" @default.
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- W58602382 doi "https://doi.org/10.1016/s0076-6879(01)29072-7" @default.
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