His‐tagged MtrA and GlnR were expressed and purified essentially as described (Zhu et al., 2019 (link)). In brief, protein production was induced by addition of 1 mM IPTG, and bacterial cells were collected after overnight culture at 16°C and then re‐suspended and sonicated in binding buffer [50 mM NaH2PO4 (pH 8.0), 200 mM NaCl, 20 mM imidazole] on ice. Crude lysates were centrifuged to remove cell debris, and soluble proteins in supernatant were purified by Ni affinity column (Qiagen, USA). Purified proteins were examined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and their concentration was determined by the Pierce BCA Protein Assay Kit (Thermo Scientific, USA).
Ni affinity column
Manufactured by Qiagen
Sourced in United States
The Ni affinity column is a laboratory equipment designed for the purification of His-tagged proteins. It utilizes nickel (Ni) bound to the solid support matrix to selectively capture and isolate the target protein from complex mixtures. The column functions by exploiting the high affinity between the histidine tag and the nickel ions, allowing for efficient separation and concentration of the protein of interest.
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Lab products found in correlation
2 protocols using ni affinity column
1
Purification of His-tagged MtrA and GlnR
2
Purification of His-tagged MtrA and GlnR
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His-tagged MtrA and GlnR were expressed and purified essentially as described (Zhu et al., 2020a , Zhu et al., 2019 , Lu et al., 2018) . In brief, protein production was induced by addition of 1 mM IPTG, and bacterial cells were collected after overnight culture at 16°C and then re-suspended and sonicated in binding buffer [50 mM NaH 2 PO 4 (pH 8.0), 200 mM NaCl, 20 mM imidazole] on ice.
Crude lysates were centrifuged to remove cell debris, and soluble proteins in supernatant were purified by Ni affinity column (Qiagen, USA). Purified proteins were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and their concentration was determined by the Pierce BCA Protein Assay Kit (Thermo Scientific, USA).
Crude lysates were centrifuged to remove cell debris, and soluble proteins in supernatant were purified by Ni affinity column (Qiagen, USA). Purified proteins were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and their concentration was determined by the Pierce BCA Protein Assay Kit (Thermo Scientific, USA).
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