Bio-Rad
Native IMAC Purification Kit
6200241
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Rab proteins constitute the largest family of monomeric small GTPases that oscillate between active (GTP- bound) and inactive (GDP- bound) conformations. The spatio-temporal activation of Rabs is controlled by the GDP-GTP exchange factors (GEFs), mediating their activation, and the GTP hydrolysis activating proteins (GAPs), terminating their activity. Rab GTPases execute diverse tasks in membrane trafficking by driving cargo collection into nascent transport vesicles, linking vesicles to cytoskeletal elements, recruiting tethers, and finally docking vesicles to the target compartment, leading to membrane fusion. The goal of this project is to characterize a specific GEF for Golgi localized Rab6 GTPase, and to analyze its effect in Rab6-mediated processes at the Golgi complex. In the present experiment, His-Rab6 was purified for downstream applications using the purification kit from Biorad. The reason for using this kit was based on prior positive experiences with Biorad products.
Recombinant protein purification
Bacterial cell lysates
His-tagged Rab6 was expressed in E coli BL21 cells by induction with 0.1 mM IPTG for 4 hr at room temp. Cells were harvested and lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 5 mM MgCl2, 0.1 mM dithiothreitol (DTT), 10 µM GDP, and 1 mM PMSF). Cellslysis was done by a single pass at 20,000 p.s.i. through an EmulsiFlex-C5 (Avestin). Samples were spun at 50,000xg for 30 min at 4 deg. Clarified samples were then used for protein purification according to the manufacturer’s instructions.
Follow the instructions from the manufacturer
His-tagged Rab6 was purified with no major contaminants
None
Easy to use
Recommended for His-tagged protein purifications
Product Name: Native IMAC Purification Kit, 1 mlSupplier Page