The proteins were expressed and purified using previously developed protocols (Boura et al. 2010 , Nemecek et al. 2013) . Briefly, the proteins were expressed in E. coli strain BL21 Star. After affinity purification on Ni-NTA resin (Machery-Nagel) the solubility tag (6xHis-GB1) was removed by TEV protease and the proteins were further purified using Superdex 200 column (GE Healthcare). Proteins were concentrated to 8 mg/ml and stored at -80 °C until needed.
Ni nta resin
Manufactured by Macherey-Nagel
Sourced in Germany
Ni-NTA resin is a chromatography medium used for the purification of recombinant proteins containing a histidine-tag. It consists of nickel-nitrilotriacetic acid (Ni-NTA) immobilized on an agarose support. The histidine-tag binds to the Ni-NTA, allowing the target protein to be selectively captured and purified from complex mixtures.
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6 protocols using ni nta resin
1
Expression and Purification of Proteins
2
Purification of Recombinant ZAP-S Protein
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Recombinant ZAP-S N-terminally tagged with 6×His-SUMO was purified from E. coli Rosetta 2 cells (Merck) by induction with 0.2 mM isopropyl β-d-1-thiogalactopyranoside for 18 h at 18 °C. Cells were collected, resuspended in lysis buffer (50 mM HEPES/KOH pH 7.6, 1 M NaCl, 1 mM DTT, 1 mM PMSF) and lysed in a pressure cell. The lysate was cleared by centrifugation and ZAP-S was captured using Ni-NTA resin (Macherey-Nagel). After elution with 500 mM imidazole, ZAP-S was further purified and the bound nucleic acids removed by size exclusion chromatography (HiLoad® 16/600 Superdex® 200) in 20 mM HEPES/KOH pH 7.6, 1 M KCl, 1 mM DTT, 20% glycerol. Protein identity was verified by SDS-PAGE as well as western blotting (Supplementary Fig. 2D ). Purified ZAP-S was rapidly frozen and stored in aliquots at −80 °C. His-SUMO IGF2BP3 as well as His-SUMO were kind gifts from Dr. Andreas Schlundt (Goethe University, Frankfurt, Germany).
3
Purification of Recombinant ZAP-S Protein
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Recombinant ZAP-S N-terminally tagged with 6×His-SUMO was puri ed from E. coli Rosetta 2 cells (Merck) by induction with 0.2 mM isopropyl β-d-1-thiogalactopyranoside for 18 h at 18°C. Cells were collected, resuspended in lysis buffer (50 mM HEPES/KOH pH 7.6, 1 M NaCl, 1 mM DTT, 1 mM PMSF) and lysed in a pressure cell. The lysate was cleared by centrifugation and ZAP-S was captured using Ni-NTA resin (Macherey-Nagel). After elution with 500 mM imidazole, ZAP-S was further puri ed and the bound nucleic acids removed by size exclusion chromatography (HiLoad® 16/600 Superdex® 200) in 20 mM HEPES/KOH pH 7.6, 1 M KCl, 1 mM DTT, 20% glycerol. Protein identity was veri ed by SDS-PAGE as well as western blotting (Supplementary Fig. 2D). Puri ed ZAP-S was rapidly frozen and stored in aliquots at -80°C.
His-SUMO-IGF2BP3 as well as His-SUMO were kind gifts from Dr. Andreas Schlundt (Goethe University, Frankfurt, Germany)
His-SUMO-IGF2BP3 as well as His-SUMO were kind gifts from Dr. Andreas Schlundt (Goethe University, Frankfurt, Germany)
4
Purification of Serine Protease ClpP
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Pellets were homogenized with buffer (A): 50 mM Tris base (pH 8.0), 100 mM NaCl, 20% w/v glycerol, or buffer (B): 50 mM Tris base (pH 8.0), 100 mM NaCl, 2 mM DTT. After sonication on ice and centrifugation, the supernatant was mixed with Ni–NTA resin (MACHEREY–NAGEL GmbH & Co. KG, Germany), previously equilibrated with (A) or (B). Protein sample eluted at 300 mM imidazole was dialyzed overnight in (A) or (B). Subsequently, SeClpP sample was submitted to size-exclusion chromatography in a HiLoad 26/600 Superdex 200 preparation grade (pg) column connected to an ÄKTA Purifier 10 system (General Electric, Sweden). The inactive mutant of SeClpP (S98A) was also expressed and purified as described in “Heterologous expression of SeClpP ” and “Cell lysis and protein purification ” sections. SeClpP S98A was applied to an enzymatic assay to confirm the absence of endogenous ClpP from Escherichia coli, as shown in Supplementary Fig. S6 .
5
Purification of ZAP-S Protein
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Recombinant ZAP-S N-terminally tagged with 6×His-SUMO was purified from E. coli Rosetta 2 cells (Merck) by induction with 0.2 mM isopropyl β-d-1-thiogalactopyranoside for 18 h at 18 °C. Cells were collected, resuspended in lysis buffer (50 mM HEPES/KOH pH 7.6, 1 M NaCl, 1 mM DTT, 1 mM PMSF) and lysed in a pressure cell. The lysate was cleared by centrifugation and ZAP-S was captured using Ni-NTA resin (Macherey-Nagel). After elution with 500 mM imidazole, ZAP-S was further purified and the bound nucleic acids removed by size exclusion chromatography (HiLoad® 16/600 Superdex® 200) in 20 mM HEPES/KOH pH 7.6, 1 M KCl, 1 mM DTT, 20% glycerol. Protein identity was verified by SDS-PAGE as well as western blotting (Supplementary Fig. 2D). Purified ZAP-S was rapidly frozen and stored in aliquots at -80 °C.
His-SUMO-IGF2BP3 as well as His-SUMO were kind gifts from Dr. Andreas Schlundt (Goethe University, Frankfurt, Germany)
His-SUMO-IGF2BP3 as well as His-SUMO were kind gifts from Dr. Andreas Schlundt (Goethe University, Frankfurt, Germany)
6
Purification and Characterization of scFv from E. coli
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E. coli HB2105 infected with selected monoclonal phages were grown at 30 °C in 2× TY medium supplement with 1% glucose until OD600 = 0.8, followed by induction overnight with 1 mM IPTG. The induced cells were harvested and the soluble scFv, localized in the periplasmic space, was extracted using BugBusterTM. The induced bacterial cell pellets from 100 mL of cell culture were re-suspended with 1 mL BugBusterTM. The suspension was centrifuged for 10 min at 12,000× g. scFvs contained in the periplasmic space were purified with Ni-NTA resin (Macherey-Nagel) as recommended by the manufacturer. The purity of the scFvs was determined by SDS-PAGE followed by a Coomassie Brilliant Blue staining. The concentration of purified scFvs was measured using Bradford reagent (Bio-Rad). The binding activity of purified scFvs to adiponectin was analyzed by ELISA.
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