The DNA sequence encoding the extracellular domain of human p75NTR (residues 1–161) with a hexahistidine tag at the C terminus was subcloned into the pFASTBac1 vector (Invitrogen). The extraction of bacmids and preparation of baculovirus were performed with the same procedure as used for rPRELP. For recombinant, soluble p75NTR (rsp75NTR) expression, 1.8 × 106 cells/ml of Sf9 cells (Thermo Fisher Scientific) were infected with P3 virus (1:1000, v/v) and incubated with shaking at 120 rpm at 27 °C for 5 days. The rsp75NTR was purified from the supernatant by a Ni-NTA agarose affinity column (Qiagen) equilibrated with binding buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM imidazole). The column was first washed with the binding buffer, and fractions were eluted with buffers containing increasing concentrations of imidazole (20–500 mM). The eluted fractions containing the rsp75NTR fragment were further purified by size-exclusion chromatography using a HiLoad 26/600 Superdex 75 pg column (GE Healthcare) equilibrated with 20 mM Tris-HCl (pH 8.0), 150 mM NaCl.
Ni nta agarose affinity column
Manufactured by Qiagen
Sourced in United Kingdom
Ni-NTA agarose affinity column is a chromatography resin used for the purification of recombinant proteins containing a histidine-tag (His-tag). The resin consists of nickel-nitrilotriacetic acid (Ni-NTA) coupled to agarose beads, which selectively binds to the His-tag on the target protein, allowing it to be separated from other cellular components during the purification process.
Automatically generated - may contain errors
Lab products found in correlation
French press, by Thermo Fisher Scientific (2 mentions)
Sf9 cells, by Thermo Fisher Scientific (1 mentions)
Hiload 26 600 superdex 75pg column, by GE Healthcare (1 mentions)
Pfastbac1 vector, by Thermo Fisher Scientific (1 mentions)
Hiload 16 600 superdex 200 pg column, by Cytiva (1 mentions)
Expifectamine 293 transfection kit, by Thermo Fisher Scientific (1 mentions)
Pcdna3.4 vector, by Thermo Fisher Scientific (1 mentions)
Expi293 cell, by Thermo Fisher Scientific (1 mentions)
Hiload 26 600 superdex 200 pg column, by GE Healthcare (1 mentions)
Expifectamine cho transfection kit, by Thermo Fisher Scientific (1 mentions)
Expicho cells, by Thermo Fisher Scientific (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Pd 10, by GE Healthcare (1 mentions)
Superdex200 10 30, by GE Healthcare (1 mentions)
Pd 10 desalting column, by Cytiva (1 mentions)
6 protocols using ni nta agarose affinity column
1
Recombinant Soluble p75NTR Expression
2
Recombinant FcγRIIIa Protein Production
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The DNA sequence
encoding the human FcγRIIIa (V158) ectodomain
(amino acid residues 1–175) with a C-terminal hexahistidine
tag was cloned into the pcDNA3.4 vector (Thermo Fisher Scientific).
Expi293 cells (Thermo Fisher Scientific) were transiently transfected
with the vectors using the ExpiFectamine 293 Transfection Kit (Thermo
Fisher Scientific) according to the manufacturer’s protocol.
The cells were cultured by rotating at 125 rpm for 5 days after transfection
at 37 °C and 8% CO2. The culture supernatant was collected
by centrifugation at 1000g for 10 min and filtration.
The collected supernatant was loaded onto a Ni-NTA agarose affinity
column (Qiagen) after equilibration with binding buffer [20 mM Tris-HCl
(pH 8.0), 500 mM NaCl, and 5 mM imidazole]. The resin capturing wild-type
(WT) FcγRIIIa was washed with binding buffer, and subsequently,
the protein was eluted with the buffers containing increasing concentrations
of imidazole (≤500 mM). WT FcγRIIIa was obtained after
further purification by size-exclusion chromatography using a HiLoad
16/600 Superdex 200 pg column (Cytiva) equilibrated with 50 mM Tris-HCl
(pH 8.0), 500 mM NaCl, and 1 mM EDTA.
encoding the human FcγRIIIa (V158) ectodomain
(amino acid residues 1–175) with a C-terminal hexahistidine
tag was cloned into the pcDNA3.4 vector (Thermo Fisher Scientific).
Expi293 cells (Thermo Fisher Scientific) were transiently transfected
with the vectors using the ExpiFectamine 293 Transfection Kit (Thermo
Fisher Scientific) according to the manufacturer’s protocol.
The cells were cultured by rotating at 125 rpm for 5 days after transfection
at 37 °C and 8% CO2. The culture supernatant was collected
by centrifugation at 1000g for 10 min and filtration.
The collected supernatant was loaded onto a Ni-NTA agarose affinity
column (Qiagen) after equilibration with binding buffer [20 mM Tris-HCl
(pH 8.0), 500 mM NaCl, and 5 mM imidazole]. The resin capturing wild-type
(WT) FcγRIIIa was washed with binding buffer, and subsequently,
the protein was eluted with the buffers containing increasing concentrations
of imidazole (≤500 mM). WT FcγRIIIa was obtained after
further purification by size-exclusion chromatography using a HiLoad
16/600 Superdex 200 pg column (Cytiva) equilibrated with 50 mM Tris-HCl
(pH 8.0), 500 mM NaCl, and 1 mM EDTA.
3
Recombinant Soluble IGFI-R Production
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The DNA sequence encoding the extracellular domain of human IGFI-R (residues 1–905) with a hexahistidine tag at the C terminus was subcloned into the pcDNA3.4 vector. ExpiCHO cells (Thermo Fisher Scientific) were transiently transfected with the vector using ExpiFectamine CHO Transfection Kit (Thermo Fisher Scientific) in accordance with the manufacturer’s max-titer protocol. The cells were cultured for 2 weeks at 32 °C and 5% CO2. The recombinant, soluble IGFI-R (rsIGFI-R) was purified from the supernatant by a Ni-NTA agarose affinity column (Qiagen) equilibrated with binding buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM imidazole). The column was first washed with the binding buffer, and fractions were eluted with buffers containing increasing concentrations of imidazole (20–500 mM). The eluted fractions containing rsIGFI-R were further purified by size-exclusion chromatography using a HiLoad 26/600 Superdex 200 pg column (GE Healthcare) equilibrated with 20 mM Tris-HCl (pH 8.0), 150 mM NaCl.
4
Recombinant Protein Purification from E. coli
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LP with an N-terminal polyhistidine tag was expressed in E. coli (JM109 (DE3)) at 23 °C for 65 h in LB bacterial growth medium supplemented with 0.1 mg ml−1 carbenicillin. Cells were collected and ruptured with a French press (Thermo Fisher Scientific), and recombinant proteins were purified from the supernatant using Ni-NTA agarose affinity columns (Qiagen) followed by buffer-exchange (20 mM HEPES, pH 7.4) gel filtration (PD-10 column, GE Healthcare). The whole purification process after rupture was conducted on ice to avoid protein degradation. The protein concentration was determined by Bradford method (Protein assay kit, Bio-Rad).
5
Recombinant Protein Purification from E. coli
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WH indicator was expressed in Escherichia coli strain JM109 (DE3), and cultured at 23 °C for 69 h in 200 mL LB bacterial growth medium supplemented with 100 µg/mL carbenicillin. Cultured cells were suspended in PBS buffer, and lysed using a French press (ThermoFisher Scientific). The lysate was centrifuged (8000 rpm at 4 °C for 20 min), and recombinant proteins were purified from the supernatant using Ni-NTA agarose affinity columns (QIAGEN), followed by buffer exchange with 20 mM HEPES (pH 7.4) with the desalting column, PD-10 (GE Healthcare, Buckinghamshire, UK). After lysis, all protein purification processes were conducted on ice to avoid protein degradation. Protein concentration was determined by both the alkaline-denaturation method [36 (link),37 (link)] and the Bradford assay.
6
Isolation and Purification of ofCP from O. formosa
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Isolation of ofCP from O. formosa was described in Supplementary Note 2. The gene encoding the CPs was inserted into the pRSETB vector, which is a plasmid vector for high protein expression with a cleavable polyhistidine tag. The plasmid was then transformed into E. coli JM109(DE3) and spread over the surface of LB agar medium with 100 μg/mL ampicillin. After overnight incubation at 37°C, single colonies were inoculated into 200 mL of LB liquid medium containing carbenicillin (100 μg/mL), and cultured with vigorous shaking in an incubator at 37°C for 69 h. The harvested bacterial cell lysate by French Press G‐M (Glen Mills Inc.) following by purification using Ni‐NTA agarose affinity columns (QIAGEN). The purified protein was eluted with 2 mL of 200 mM imidazole buffer. A PD‐10 desalting column (Cytiva) was used for buffer exchange of purified protein to 20 mM HEPES at a pH of 7.4. Size‐exclusion chromatography (SEC), using ÄKTA (GE Healthcare) equipped with a Superdex200 10/30 (GE Healthcare) column, was used for further purification. Filtration using a 0.2 μm pore filter was conducted in advance to remove the impurities before loading the proteins onto the column. The same procedure was followed to identify the oligomerization status. Photophysical analysis was described in Supplementary Note 3.
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