Recombinant GST-KHK-A, GST-YWHAH, and free GST proteins were expressed in Escherichia coli BL21 cells, pulled down using glutathione-affinity beads (GE Healthcare; Chicago, IL) at 4 °C for 1 h, and eluted with 10 mM reduced glutathione (Sigma-Aldrich). FLAG-LRRC59 and His(6)-KHK-A proteins were expressed in HEK293T cells, pulled down using EZview Red anti-FLAG (Sigma-Aldrich) and nickel-NTA (Qiagen, Hilden, Germany) affinity beads at 4 °C for 4 h, and eluted with 500 ng/μL of FLAG peptide and 250 mM imidazole, respectively. The amounts and purities of proteins were checked by SDS-PAGE and Coomassie Brilliant Blue R-250 staining.
Glutathione affinity beads
Manufactured by GE Healthcare
Glutathione-affinity beads are a type of chromatography resin used for the purification of glutathione S-transferase (GST)-tagged recombinant proteins. The beads are composed of agarose or polymer matrices that are covalently linked to glutathione, a tripeptide that binds to the GST tag on the target protein. This allows for the selective capture and isolation of the GST-tagged protein from complex mixtures, such as cell lysates or culture supernatants.
Automatically generated - may contain errors
2 protocols using glutathione affinity beads
1
Affinity Purification of Recombinant Proteins
2
Affinity Purification of Recombinant Proteins
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Recombinant GST-FIH, GST-ODDD, GST-CT, and free GST proteins were expressed in Escherichia coli BL21 cells, pulled down using glutathione-affinity beads (GE Healthcare; Chicago, IL) at 4 °C for 1 h, and eluted with 10 mM reduced glutathione (Sigma-Aldrich). Recombinant His(6)-NAA10 WT and W38F proteins were expressed in BL21 cells, bound to Nickel-NTA affinity beads (Qiagen; Hilden, Germany) at 4 °C for 1 h, and eluted with 250 mM imidazole. The amounts and purities of extracted proteins were checked by SDS-PAGE and Coomassie Brilliant Blue R-250 staining.
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