CtBP1 was subcloned into the pET28a vector and expressed in the BL21(DE3) E. Coli strain (Novagen, Darmstadt, Germany) and purified from the bacterial lysate using Ni-Sepharose HP resin (Amersham Biosciences, Amersham, UK). Eluate from the Ni resin was further purified on a Superdex 200 size exclusion column (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Adenovirus 5 E1A was subcloned into the pGEX-KG vector and transformed into DH5α E. Coli strain (Life Technologies, Carlsbad, CA). GST-fused E1A was first purified using Glutathione Sepharose 4B resin (GE Healthcare), then on a Superdex 200 size exclusion column (GE Healthcare). Both purified proteins were concentrated, aliquoted, and stored at −80 °C in lysis buffer (100 mM Tris, pH 8.0, 250 mM NaCl, 5% glycerol, and 1 mM dithiothreitol).
Ni sepharose hp resin
Manufactured by Cytiva
Sourced in United Kingdom
Ni-Sepharose HP resin is a high-performance affinity chromatography resin designed for the purification of histidine-tagged proteins. It consists of nickel-charged iminodiacetic acid (IDA) ligands covalently coupled to highly cross-linked agarose beads. The resin offers high binding capacity and selectivity for histidine-tagged proteins, enabling efficient capture and purification.
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2 protocols using ni sepharose hp resin
1
Purification of CtBP1 and Adenovirus 5 E1A Proteins
2
Purification of Recombinant Histidine-tagged ApoA1
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The bacterial expression vector encoding codon-optimized his-tagged human apoA1 has been previously described [17 (link)]. All point mutations and deletions were created using the QuickChange II Mutagenesis Kit (Thermo Fisher). All mutations were confirmed by DNA sequencing. Expression plasmids were transformed into E. coli BL21 dE3 pLysS and protein expression was induced in shaking cultures by overnight incubation with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside at room temperature. The resulting cellular pellet was resuspended in B-PER lysis solution (Thermo Fisher) containing Lysozyme, DNaseI, and a protease inhibitor cocktail. The cellular debris was removed by centrifugation and the supernatant was diluted into PBS containing 3 M guanidine-HCl. The denatured histidine-tagged apoA1 was purified using Ni Sepharose HP resin (Amersham Biosciences) followed by imidazole elution. Fractions containing recombinant apoA1 were extensively dialyzed against PBS and analyzed for purity by SDS-PAGE and Coomassie Blue staining. Only samples with >95% purity were used. When indicated apoA1 was reduced in 10 mM DTT, and reductive methylation was performed with 100 mM N-ethylmaleimide (NEM) at 37°C for 1 hr under nitrogen gas, followed by dialysis in PBS.
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