The DENN domain of Sbf or full-length Rab35 or Rab11 was amplified by PCR and cloned in-frame into pET-15b vector. The recombinant proteins were induced for 16 h at 20°C in Escherichia coli BL21 (DE3) using 1 mM IPTG. The bacteria were lysed by sonication and 6xHis- or MBP-tagged fusions were purified by incubation with Cobalt resin beads (catalog no. 89964; Thermo Fisher Scientific) or amylose resin beads (catalog no. E8021S; New England Biolabs), respectively. The recombinant proteins were eluted with 250 mM imidazole or 20 mM maltose in PBS buffer. Pull-down assays were performed by incubating 50 µg MBP fusions prebound to amylose resin beads for 1 h at room temperature with 6xHis fusions protein.
Cobalt resin beads
Manufactured by Thermo Fisher Scientific
Cobalt resin beads are a type of affinity chromatography media used for the purification of proteins and other biomolecules. They consist of cross-linked agarose beads functionalized with cobalt ions, which can selectively bind to proteins with specific histidine-containing tags. The core function of cobalt resin beads is to facilitate the isolation and purification of tagged proteins from complex mixtures.
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2 protocols using cobalt resin beads
1
Purification and Pull-down Assay of Recombinant Proteins
2
Recombinant MuSK Tetramer Production
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The extracellular domain of human MuSK was subcloned into the pMT/Bip/His-A vector. The C-terminal region contained a short, flexible linker followed by a BirA site (amino acids: GLNDIFEAQKIEWHE), downstream of which was a thrombin-cleavable (amino acids: LVPRGS) 6× histidine tag. Protein expression was induced in S2 Drosophila cells (Thermo Fisher Scientific). Culture supernatant was collected and MuSK protein was purified using cobalt resin beads (Thermo Fisher Scientific) according to the manufacturer’s instructions. For tetramer formation, MuSK protein was biotinylated by incubation with BirA enzyme at a 1:100 molar ratio overnight at 4°C in a buffer containing 50 mM Tris, 50 mM bicine at pH 8.3, 10 mM magnesium acetate, 10 mM adenosine-5’-triphosphate, and 50 μM biotin. Excess biotin was removed using a 10-kDa MWCO Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific). Fluorescent multimers were formed using stepwise addition of APC-conjugated streptavidin (Invitrogen) to biotinylated MuSK until a 1:4 molar ratio was reached.
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