Recombinant PbICL protein was obtained as described by Cruz [14] (link). Briefly, Pbicl cDNA was inserted into the pET-32a (+) expression vector (Novagen, Inc,). The resulting plasmid was transformed into E. coli BL21 C43 (DE3) cells, and expression was induced at an A600 of 0.6 by the addition of 1 mm (final concentration) isopropyl thio-β-D-galactoside (IPTG) (Sigma-Aldrich). After induction, the cells were incubated for another 2 h at 36°C with shaking at 200 rpm. The cells were harvested by centrifugation at 10,000×g for 5 min at 4°C and resuspended in 1 × NaCl/Pi buffer. After incubation for 30 min with 100 µg/mL lysozyme, the cells were lysed by extensive sonication. The sample was centrifuged at 4°C and 8,000×g for 15 min, and the supernatant, which contained the soluble protein fraction, was collected. His-tagged ICL was purified using the Ni-NTA Spin Kit (Qiagen), and the tags were subsequently removed by the addition of EKMax™ enterokinase (Invitrogen).
Ni nta spin kit
Manufactured by Qiagen
Sourced in Germany, United States
The Ni-NTA Spin Kit is a column-based purification system designed for the efficient purification of His-tagged proteins from a variety of sample types. The kit utilizes Ni-NTA agarose resin to capture and purify the target protein, which can then be eluted using an imidazole-containing buffer. The spin column format allows for rapid and convenient processing of multiple samples simultaneously.
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Lab products found in correlation
Adenosine triphosphate (atp), by Roche (2 mentions)
Takara bca protein assay kit, by Takara Bio (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Isopropylthio β d galactoside iptg, by Merck Group (1 mentions)
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (1 mentions)
Pierce 660 nm protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx stain free precast gel, by Bio-Rad (1 mentions)
Jm109, by Qiagen (1 mentions)
T4 dna ligase, by Promega (1 mentions)
Restriction enzyme, by Thermo Fisher Scientific (1 mentions)
Genejet pcr purification kit, by Thermo Fisher Scientific (1 mentions)
Genejet gel extraction kit, by Thermo Fisher Scientific (1 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions)
Dreamtaq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Akta fplc machine, by GE Healthcare (1 mentions)
Ni nta resin, by Qiagen (1 mentions)
Streptavidin sepharose high performance, by GE Healthcare (1 mentions)
Bl21 de3 plyss, by Agilent Technologies (1 mentions)
Pgex 6p 1 vector, by Cytiva (1 mentions)
31 protocols using ni nta spin kit
1
Recombinant PbICL Protein Purification
2
Characterization of PdMYB221 Transcription Factor
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The PdMYB221 coding region was fused in frame with HIS in the pET28a vector and expressed in Escherichia coli. The recombinant protein was purified using Ni-NTA Spin Kit (QIAGEN, Germany). The PdCESA8, PdGT47C, and PdCOMT2 promoter fragments used as the probes were amplified with primers labeled with biotin at the 5′ end (Table S2 ). The EMSA assay was conducted with a LightShift® Chemiluminescent EMSA Kit (Thermo Fisher) according to the manufacturer’s instructions. Briefly, the labeled DNA fragments were incubated for 30 min with 100 ng of the recombinant protein in binding buffer (10 mM Tris, pH 7.5, 50 mM KCL, 1 mM DTT, 2.5% glycerol, 5 mM MgCl2, 0.05% Nonidet P-40 and 100 ng/μl poly (dl-dC)). The PdMYB221-bound DNA fragments were separated from the unbound fragments by polyacrylamide gel electrophoresis. The DNA was electroblotted onto a nitrocellulose membrane and detected by chemiluminescence.
3
Purification and kinetic analysis of DapF variants
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The dapF variants were PCR amplified from boiled cells that contained the desired mutation (wild‐type E. coli MG1655 for the wild‐type dapF sequence; reconstructed dapF mutants for the G210D and M260Y variants). The PCR products were then cloned and sequence verified into a custom‐made pET‐3 backbone, containing the histidine tag (6×) on either the 5′ or 3′ end of the genes to test for optimal expression. Corynebacterium glutamicum DAP dehydrogenase was synthesized from Eurofins Genomics and also cloned in the pET‐based vector. Expression was done in a E. coli BL21 strain using LB media, which was induced with 1 mM IPTG when OD600 reached 0.6. Induced cultures were grown at 30°C overnight under 200 rpm, harvested by centrifugation, and the pellet stored at −80°C for protein purification.
Proteins were purified using the Ni‐NTA Spin Kit (QIAGEN), following the protocol for purification of tagged proteins under native conditions. Purified samples were run on a denaturing PAGE gel (Mini‐PROTEAN TGX Stain‐Free Precast Gels, Bio‐Rad) to confirm purity and quantified using the Thermo Fisher Scientific Pierce 660 nm Protein Assay Reagent. Purified proteins were used fresh for the kinetic assay (never frozen).
Proteins were purified using the Ni‐NTA Spin Kit (QIAGEN), following the protocol for purification of tagged proteins under native conditions. Purified samples were run on a denaturing PAGE gel (Mini‐PROTEAN TGX Stain‐Free Precast Gels, Bio‐Rad) to confirm purity and quantified using the Thermo Fisher Scientific Pierce 660 nm Protein Assay Reagent. Purified proteins were used fresh for the kinetic assay (never frozen).
4
Cloning and Expression of Pre-miniproAb
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The expression vector pET-24a(+) was obtained from Novagen and the pMA-PMPA plasmid (a pMA plasmid harboring Pre-miniproAbollien gene) from Geneart, Germany. Restriction enzymes, T4 DNA ligase, kits for DNA gel extraction and purification and kits for plasmid extraction and purification were purchased from Promega, USA. Protein purification Ni–NTA Spin Kit was purchased from Qiagen, Germany. E. coli host strains JM109 (Genotype: endA1, recA1, gyrA96, thi, hsdR17 (rk−, mk+), relA1, supE44, Δ(lac-proAB), [F′ traD36, proAB, laqIqZΔM15]), JM109(DE3) (Genotype: endA1, recA1, gyrA96, thi, hsdR17 (rk−, mk+), relA1, supE44, λ−, Δ(lac-proAB), [F′, traD36, proAB, lacIqZΔM15], λDE3) and BL21(DE3)pLysS (Genotype: F−, ompT, hsdSB (rB−, mB−), dcm, gal, λ(DE3), pLysS, Cmr) were obtained from Qiagen, Germany. Designed gene was synthesized and optimized in Geneart, Germany. Forward and reverse T7 promoter primer and T7 terminator primer were synthesized by Operon Company, France.
5
Quantifying CypB Binding Kinetics
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Ten microliters of His-CypB (2.5 µM, 2 µM, 1.5 µM, and 1 µM) was incubated with 10 µL of settled Ni-NTA macroporous silica (Ni-NTA Spin Kit; Qiagen) for 30 min at 0 °C. Subsequently, 80 µL of HEPES buffer was added and incubated for another 10 min with gentle shaking. The silica was precipitated by gravity for 10 min. Eighty microliters were taken from the supernatant and measured along with the silica fraction (the remaining 20 µL) for PPIase activity. The amounts of CypB in the wells were 25, 20, 15, and 10 pmol. Additionally, the activity of 10 µL of 100 nM His-CypB in solution was measured. Three independent experiments were performed. The result is shown in Supplementary Fig. 9 .
6
Molecular Protocols for Erwinia amylovora Screening and Purification
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Primers (see Additional file 6 ) were designed based on E. amylovora CFBP1430 genome sequences available from NCBI (GenBank NC_013961.1). Screening PCR reactions were carried out using the DreamTaq DNA polymerase (Thermo Scientific) in accordance with the manufacturer’s instructions and optimized annealing temperatures based on the melting temperatures of the respective primers. For high fidelity PCR reactions, Phusion DNA polymerase (Thermo Scientific) was used where the annealing temperature was 3°C higher than the lower temperature of the used primer combination.
Restriction enzyme (Thermo Scientific) and T4 DNA ligase (Thermo Scientific) reactions were performed as per the manufacturer’s instructions at the appropriate temperature where all ligation reactions were incubated at room temperature.
DNA purifications were either performed using the GeneJET PCR purification kit (Thermo Scientific) or the GeneJET Gel extraction kit (Thermo Scientific) following the manufacturer’s instructions.
Protein purification was carried out using the Ni-NTA Spin Kit (Qiagen) following the manufacturer’s instructions.
Restriction enzyme (Thermo Scientific) and T4 DNA ligase (Thermo Scientific) reactions were performed as per the manufacturer’s instructions at the appropriate temperature where all ligation reactions were incubated at room temperature.
DNA purifications were either performed using the GeneJET PCR purification kit (Thermo Scientific) or the GeneJET Gel extraction kit (Thermo Scientific) following the manufacturer’s instructions.
Protein purification was carried out using the Ni-NTA Spin Kit (Qiagen) following the manufacturer’s instructions.
7
Purification of Hexahistidine-Tagged AspBHI Protein
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Wild type (WT) and mutant AspBHI with N-terminal 6xHis tags were expressed in a Dcm-deficient E. coli strain T7 Express (C2566). Cells were grown at 30°C in 10 mL (small scale) or 0.5 to 1 L (medium scale) in LB + Amp to OD600 0.3–0.6 and induced with a final concentration of 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). Induced cultures were grown overnight at 25°C, harvested and then kept at −20°C. His-tagged proteins (small scale) were partially purified using Qiagen Ni-NTA spin kit as recommended by the supplier and used in the experiments shown in Fig. 4 . For medium-scale production cells were lysed using sonication in 20 mM Tris-HCl, pH 7.5, 400 mM NaCl, 20 mM imidazole. Clarified cell extract was loaded over a gravity column using a Ni-NTA resin (Qiagen). Protein was eluted with 500 mM imidazole. Pooled fractions were then diluted by 10 fold in 20 mM Tris-HCl, pH 7.5, 20 mM NaCl and loaded over a 5 mL Hi-Trap Heparin column using an AKTA FPLC machine (GE Healthcare). The proteins were eluted at ~250–290 mM NaCl with a linear gradient of 20 mM to 1 M NaCl. Fractions containing AspBHI were identified on 10–20% gradient Tris-Glycine gels (Novex/Life Technologies) with the protein appearing as the major band (purity approximately 95%; Fig. 5b insert). Proteins were diluted to a working stock of 0.5–1 mg ml−1 and used in the experiments shown in Fig. 5b .
8
Site-directed Mutagenesis of AlfC α1,6-Fucosidase
9
SNX27 and FAM21 Protein Interaction
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SNX27 cDNA was subcloned into pGEX-6P-1 vector (Amersham) to be fused with GST, and FAM21 cDNA was subcloned into pRSET-A vector (Invitrogen) to be fused with polyhistidine (6 × His). Plasmids were transformed into E. coli strain BL21 (DE3) pLysS (Agilent). E. coli were grown and induced with 0.2 mM isopropyl-β-D -thiogalactopyranoside at 30 °C for 3 h. Bacterial cells were collected and proteins were purified with Glutathione Sepharose 4B beads (GST fusions; GE Healthcare) or Ni-NTA spin kit (polyhistidine fusions; Qiagen), as per the manufacturer's instructions. After binding, beads were washed three times with binding buffer, and boiled with SDS–PAGE sample buffer. For peptide pull-down assays, peptides corresponding to residues 40–79 (DAGLLQFLQEFSQQTISRTHEIKKQVDGLIRETKATDCRL) and 592–600 (TLCLQAQRE) of FAM21 were synthesized and biotinylated at the N-terminus (GenScript). 1 μg of peptides and 1 μg of bacterially purified CBP-tagged SNX27 proteins were incubated in binding buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Triton X-100) at 4 °C for 16 h, followed by incubation with Streptavidin Sepharose High Performance (GE Healthcare) at 4 °C for an additional 1 h. Beads were washed three times with binding buffer, and bound proteins were analysed by immunoblotting.
10
Synthesis and Characterization of Gold Nanoparticles
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HAuCl4·3H2O (99.9%), NaBH4 (99%), ascorbic acid (99%), hexadecyl trimethyl ammonium bromide (CTAB) (99%), ascorbic acid (99%), AgNO3 (99%), and bovine serum albumin (BSA) were purchased from Sigma Aldirich (St. Louis, MO, USA). Lactose and trisodium citrate were purchased from Merck (Darmstadt, Germany), Isopropyl d-thiogalactopyranoside (IPTG) Takara (Shiga, Japan), ATP from Roche (Basel, Switzerland), coelenterazine and D-luciferin potassium salt from Resem (Lijnden, Netherlands). The Ni-NTA spin kit was provided from Qiagen Inc (Hilden, Germany). Deionized water (Millipore Milli-Q grade) with a resistivity of 18.2 MΩ·cm was used in all experiments. Glassware was thoroughly cleaned with aqua regia and rinsed with DI water.
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