Recombinant CyaY, IscUWT and IscUIM proteins containing a C-terminal His6 tag were expressed in E. coli and purified as follows: E. coli BL21 (DE3)/pETcyaY was grown in LB medium containing 50 μg/mL ampicillin at 37°C. Protein expression was induced for 4 h by the addition of 0.5 mM isopropyl β-D-thiogalactoside (IPTG) at an OD600 ≈ 0.5. The bacterial pellet was resuspended in buffer A (0.1 M Tris-HCl, pH 8, 500 mM NaCl, 20 mM imidazole) and disrupted in a French press. After centrifugation (15 min, 11 000 rpm, 4°C), the supernatant was loaded onto a 1-mL HisTrap affinity column (GE Healthcare) equilibrated with buffer A. Proteins were eluted with a gradient of buffer A containing 500 mM imidazole. Protein-containing fractions were desalted with a Nap-25 column (Amersham Biosciences) and then concentrated. A similar procedure was used to purify IscUWT and IscUIM proteins except that protein expression was induced by the addition of 1 mM IPTG. Recombinant E. coli IscS containing an N-terminal His6 tag was expressed and purified as previously described [74 (link)]. The protein concentration was estimated by measuring the absorbance at 280 nm with the NanoDrop2000 spectrophotometer and using the calculated molar extinction coefficient.
Nap 25 column
Manufactured by Cytiva
Sourced in Sweden
The Nap-25 column is a size-exclusion chromatography (SEC) column designed for the separation and purification of biomolecules such as proteins, peptides, and other macromolecules. The column features a 25 mm diameter and is suitable for small-scale, analytical, and preparative applications.
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Lab products found in correlation
4 protocols using nap 25 column
1
Purification of Fe-S Cluster Proteins
2
Purification of Recombinant MtTPS5 Enzyme
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Strains of E. coli (BL21-CodonPlus(DE3)) with recombinant vectors of MtTPS5 12 and N-terminal His 8 -tag were grown to A 600 = 0.5 at 37 °C in LB-medium with kanamycin (50 μg ml -1 ). After induction with isopropyl β-D-1-thiogalactopyranoside (IPTG), cultures were shaken overnight at 16 °C. Cells were harvested by centrifugation and the pellet was resuspended in lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0) and incubated with lysozyme. After disruption of the cells by sonication, cell debris were removed by centrifugation. The supernatant was passed over a column of Ni 2+ -NTA-Agarose (QIAGEN, Germany), equilibrated with lysis buffer. After being washed twice with washing buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM imidazole), the protein was eluted with elution buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole). The purified protein was desalted into a TRIS-buffer (50 mM TRIS, pH 7.5, 10 mM NaCl, 10% glycerol) by passing through a NAP 25 column (Amersham Biosciences, Sweden), diluted to reach a concentration of 0.2 and 1 mg ml -1 and stored at -20 °C.
3
Recombinant DJ-1 Protein Expression and Purification
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The pET3a-His-DJ-1
plasmid was a gift from the Michael J Fox Foundation MJFF (Addgene
plasmid #51488). The C106A mutant was generated using Agilent QuikChange
Site-Directed Mutagenesis with the pET3a-His-DJ-1 plasmid as a template
and the following reported primers, as previously reported:17 (link)5′-GCCGCCATTGCCGCAGGCCCGACCGC-3′
5′-AATCAGGCCTTTGCGGTTCTCCTGCTCTTTCAGG-3′
Both
DJ-1WT and DJ-1C106A proteins were expressed
in BL21(DE3) E. coli (Agilent). Cultures
were grown to an OD600 value of 0.6 at 37 °C and induced
overnight at 18 °C using IPTG. Cells were then pelleted and frozen.
Frozen pellets were thawed and then lysed with 50 mM Tris-HCl pH 7.0,
500 mM NaCl, 1 mM MgCl, 40 μM Benzonase (Millipore Sigma), and
0.4 mM PMSF via sonication on ice for 3.5 min. Lysates
were loaded onto a Ni-NTA column (Cytiva) with 20 mM PBS pH 7.4, 20
mM imidazole, and 500 mM NaCl. His-tagged protein was eluted using
20 mM PBS pH 7.4, 500 mM imidazole, and 500 mM NaCl before being buffer-exchanged
into 20 mM PBS pH 7.4 using a Nap25 column (Cytiva). Protein concentrations
were determined using a Thermo Fisher Pierce BCA Protein Assay Kit
and a Tecan 10M microplate reader.
plasmid was a gift from the Michael J Fox Foundation MJFF (Addgene
plasmid #51488). The C106A mutant was generated using Agilent QuikChange
Site-Directed Mutagenesis with the pET3a-His-DJ-1 plasmid as a template
and the following reported primers, as previously reported:17 (link)5′-GCCGCCATTGCCGCAGGCCCGACCGC-3′
5′-AATCAGGCCTTTGCGGTTCTCCTGCTCTTTCAGG-3′
Both
DJ-1WT and DJ-1C106A proteins were expressed
in BL21(DE3) E. coli (Agilent). Cultures
were grown to an OD600 value of 0.6 at 37 °C and induced
overnight at 18 °C using IPTG. Cells were then pelleted and frozen.
Frozen pellets were thawed and then lysed with 50 mM Tris-HCl pH 7.0,
500 mM NaCl, 1 mM MgCl, 40 μM Benzonase (Millipore Sigma), and
0.4 mM PMSF via sonication on ice for 3.5 min. Lysates
were loaded onto a Ni-NTA column (Cytiva) with 20 mM PBS pH 7.4, 20
mM imidazole, and 500 mM NaCl. His-tagged protein was eluted using
20 mM PBS pH 7.4, 500 mM imidazole, and 500 mM NaCl before being buffer-exchanged
into 20 mM PBS pH 7.4 using a Nap25 column (Cytiva). Protein concentrations
were determined using a Thermo Fisher Pierce BCA Protein Assay Kit
and a Tecan 10M microplate reader.
4
Reconstitution of SufU Iron-Sulfur Cluster
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All steps were performed under anaerobic conditions inside a Jacomex glovebox (<1 ppm oxygen). SufU (Apo-form or Zinc-bound form, 56 µM) was pretreated anaerobically with 5 mM DTT in buffer J before incubation with 0.5 µM 6xHis-SufS and 500 µM L-cysteine for 30 min. Then, 50 mM of ammonium iron (II) sulfate hexahydrate was added, and UV-visible spectra (250–800 nm) were continuously recorded on a UVIKON XL spectrophotometer to monitor cluster formation. Unbound iron and sulfide were removed by passage on a NAP-25 column (Cytiva). The 6xHis-SufS was removed by loading the mixture onto a nickel nitrilotriacetic acid (Ni-NTA) column equilibrated with buffer J, and reconstituted SufU was recovered in the flow-through (the 6xHis-tag was cleaved before reconstitution). The amount of Fe and S content was determined using the Fish and Beinert methods, respectively [31 (link),32 (link)].
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