The cDNA for the functional upstream domain of F1 adhesin, a gift originally from Dr. Deane Mosher (University of Wisconsin, Madison, WI), was cloned into a pET15b HisTag expression vector that also contained the Maltose Binding Protein by Dr. Tomoo Ohashi and Dr. Harold Erickson (Duke University, Durham, NC), who graciously gifted the resulting MBP-FUD plasmid. This plasmid was transformed in C41 competent Escherichia coli cells and grown on ampicillin-resistant LB agar plates overnight. Individual colonies were selected and amplified in LB containing 0.1 mg/ml ampicillin. Cultures were grown to an A600 of 0.5, at which point protein expression was induced with 0.5 mM IPTG (Sigma-Aldrich, St. Louis, MO). Cultures were incubated overnight at room temperature to facilitate protein expression. Poly-histidine-tagged protein was purified from bacterial lysates by running over a cobalt resin column (Thermo Scientific, Waltham, MA) using standard protein purification techniques. Protein was eluted from the column with 0.2 M imidazole, and subsequently exchanged using a PD10 buffer exchange column (GE Healthcare Sciences, Buckinghamshire, UK). Protein concentration was quantified on a Nanodrop Spectrophotometer (Thermo Scientific, Waltham, MA).
Cobalt resin column
Manufactured by Thermo Fisher Scientific
The Cobalt resin column is a laboratory equipment designed for the purification and separation of proteins and other biomolecules. The column features a cobalt-based resin that facilitates the capture and elution of target analytes. The core function of the Cobalt resin column is to provide a reliable and efficient platform for protein purification and separation processes in a research or analytical laboratory setting.
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2 protocols using cobalt resin column
1
Purification of MBP-FUD Fusion Protein
2
Purification of His-tagged Proteins from Sf21 Cells
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Sf21 cells were infected with the baculovirus at MOI of 2. After 48 h post-infection, cells were harvested by centrifugation at 1000 g for 3 min, gently washed with resuspension buffer (20 mM HEPES pH 7.4, 0.5 M NaCl, 250 mM sucrose, protease inhibitors [5 µg/ml aprotinin, 5 µg/ml leupeptin, 2 µM pepstatin A, 1mM PMSF]). Cells were resuspended with sucrose-free resuspension buffer and then lysed by sonication on ice. After sonication, cell lysate was centrifuged at 11,000 rpm for 20 min prior to being supplemented with 0.05% Triton X-100 and then loaded onto a cobalt resin column (Thermo Scientific) pre-equilibrited with wash buffer (20 mM HEPES pH 7.4, 0.5 M NaCl, 15 mM imidazole, 0.05% TX-100). The column was washed with at least 20 column-volumes of wash buffer before elution with elution buffer (20mM HEPES pH 7.4, 0.5 M NaCl, 150 mM imidazole, 0.05% TX-100) and 0.5 ml fractions were collected and checked for protein concentration prior to being pooled and dialyzed extensively against 20mM HEPES (pH 7.4), 100 mM NaCl at 4 C.
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