The DNA plasmids encoding for each affibody were cloned into pET28, a bacterial expression vector. The vector includes the affibody gene with either only C-terminal His6-tag or biotin-acceptor peptide tag (BAP tag, GLNDIFEAQKIEW) followed by His6-tag between the NcoI and XhoI sites of pET28b (Novagen). To express affibody monomers, the vector was transformed into E. coli BL21 (DE3), and the cells were grown at 37°C in TB medium supplemented with 50mg/l kanamycin. At 0.6 OD600, 0.5mM isopropyl-β-D-thiogalactoside (IPTG) was added to induce protein expression and the cell culture was incubated for overnight at 30°C before harvest. The proteins were purified by Ni2+-NTA agarose column chromatography (Ni-NTA, Qiagen) followed by size-exclusion chromatography with a Superdex S75 10/300GL Increase column (GE Healthcare). The proteins were stored in HEPES buffered saline (HBS, 20mM HEPES pH 7.5, 150mM sodium chloride). Affibody proteins used for surface plasmon resonance experiments were site-specifically biotinylated at the C-terminal BAP tag using BirA ligase and re-purified by size-exclusion chromatography.
Superdex s75 10 300gl increase column
Manufactured by GE Healthcare
The Superdex S75 10/300GL Increase column is a size exclusion chromatography column designed for the separation and purification of proteins, peptides, and other biomolecules. It features a bed volume of 24 mL and a column dimension of 10 x 300 mm. The column packing material is composed of highly cross-linked agarose beads, which provide efficient separation of molecules based on their size and shape.
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2 protocols using superdex s75 10 300gl increase column
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Affibody Protein Expression and Purification
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Affibody Protein Expression and Purification
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The DNA plasmids encoding for each affibody were cloned into pET28, a bacterial expression vector. The vector includes the affibody gene with either only C-terminal His6-tag or biotin-acceptor peptide tag (BAP tag, GLNDIFEAQKIEW) followed by His6-tag between the NcoI and XhoI sites of pET28b (Novagen). To express affibody monomers, the vector was transformed into E. coli BL21 (DE3), and the cells were grown at 37°C in TB medium supplemented with 50mg/l kanamycin. At 0.6 OD600, 0.5mM isopropyl-β-D-thiogalactoside (IPTG) was added to induce protein expression and the cell culture was incubated for overnight at 30°C before harvest. The proteins were purified by Ni2+-NTA agarose column chromatography (Ni-NTA, Qiagen) followed by size-exclusion chromatography with a Superdex S75 10/300GL Increase column (GE Healthcare). The proteins were stored in HEPES buffered saline (HBS, 20mM HEPES pH 7.5, 150mM sodium chloride). Affibody proteins used for surface plasmon resonance experiments were site-specifically biotinylated at the C-terminal BAP tag using BirA ligase and re-purified by size-exclusion chromatography.
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