rHpl was expressed from the pET28 plasmid previously described (17 (link)) encoding a 6xHis-tagged protein. A typical expression comprised 4 L of Luria broth (Difco) at 37°C with kanamycin selection and induced at 0.6 OD600 with isopropyl b-D-1-thiogalactopyranoside (Life Technologies) for 3–4 hours. Soluble protein was lysed via sonication and applied to a 20-mL column of Ni-affinity resin (Sigma) in Ni-A buffer (50 mM l Na2HPO4, pH 7, 500 mM NaCl, 10 mM imidazole) and eluted with Ni-B buffer (50 mM l Na2HPO4, pH 7, 500 mM NaCl, 400 mM imidazole). Elutions were concentrated using a 10-kDa molecular weight cut off (MWCO) filter (ThermoFisher Scientific) to ~3 mL for further size exclusion chromatography separation using a Superdex-75 column (Cytiva, 120 mL total bed volume) in final buffer (50 mM HEPES, pH 7, 150 mM NaCl). rHpl was quantified by ultraviolet-visible spectrophotometry at 280 nm with extinction coefficient calculation (53 (link)). Fractions comprising rHpl were concentrated and stored at −80°C until further use.
Ni affinity resin
Manufactured by Merck Group
Ni-affinity resin is a chromatographic medium used for the purification of histidine-tagged proteins. It consists of nickel-charged resin that selectively binds to the histidine tag, allowing the target protein to be separated from other components in a sample.
Automatically generated - may contain errors
4 protocols using ni affinity resin
1
Purification of 6xHis-tagged rHpl Protein
2
Recombinant IL-38 Protein Purification
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A recombinant expression plasmid was ordered from Twist Bioscience (San Francisco, CA) encoding a 6xHis-tagged Small Ubiquitin-Like Modifier (SUMO) followed by mouse IL-38 residues 3 to 152 within pET21b. A typical expression comprised 4 L of luria broth at 37 °C with ampicillin selection and induced at 0.6 ODs (600 nM) with isopropyl b-D-1-thiogalactopyranoside for 3 to 4 h. Soluble protein was lysed via sonication and applied to a Ni-affinity resin (Sigma) in Ni-A buffer (50 mM l Na2HPO4, pH 7, 500 mM NaCl, 10 mM imidazole) and eluted with Ni-B buffer (50 mM l Na2HPO4, pH 7, 500 mM NaCl, 400 mM imidazole). Elutions were dialyzed against Ni-A buffer for subsequent cleavage of the 6xHis-tagged SUMO via recombinant SUMO Protease produced in-house (also known as Ulp1p, UniProt accession A0A0L8VFW2). 6xHis-tagged Sumo was stripped via Ni-affinity, and the untagged IL-38 was concentrated for size-exclusion chromatography using a Superdex-75 column (Cytiva, 120 mL total bed volume) in final buffer (50 mM HEPES, pH 7, 150 mM NaCl). Fractions comprising IL-38 were concentrated and stored at −80 °C until further use.
3
Purification and Interaction of Recombinant Proteins
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The GST alone, GST-tagged and His-tagged proteins were purified from E.coli BL21 (DE3) system. The GST-tagged proteins were enriched by Glutathione-Sepharose 4B beads (Amersham Biosciences) according to the manufacturer's instructions (Amersham Biosciences, Buckinghamshire, UK). His-tagged proteins were prepared and purified using Ni-affinity resins (Merk). His-PIM2 protein was mixed with GST or GST-HIF-1α (575-826aa) fusion proteins in PBS binding buffer (Takara's PBS, pH 7.4) at 4°C for 2 h, followed by the addition of 20 µl of Glutathione-Sepharose 4B beads. After 1h of mixing, the beads were washed with PBS five times. The pulled proteins were detected by western blots as previously described [22] (link), [23] (link).
4
Affinity Purification of Protein Complexes
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The GST alone, GST-tagged and His-tagged proteins were purified from E.coli BL21 (DE3) system. The GST-tagged proteins were enriched by Glutathione-Sepharose 4B beads (Amersham Biosciences) according to the manufacturer’s instructions (Amersham Biosciences). His-tagged proteins were prepared and purified using Ni-affinity resins (Merk). His-TTP protein was mixed with GST or GST-PKM2 fusion proteins in PBS binding buffer (Takara’s PBS, pH 7.4) at 4 °C for 2 h, followed by the additional 20 ml of Glutathione-Sepharose 4B beads. After 1 h incubation, beads were washed by PBST five times. Proteins pulled-down were detected by western blots as previously described41 .
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