The pET46 vectors (Merck Millipore; Billerica, MA, USA) encoding for the wild-type and mutant Sfβgly were previously described [25 (link)]. Recombinant enzymes were produced in NovaBlue (DE3) bacteria using the previously described protocol [25 (link)]. The enzyme purification was performed using nickel-nitrilotriacetic acid resin (Qiagen, Valencia, CA, USA) [25 (link)]. Samples of the purified enzymes were submitted to buffer exchange using HiTrap desalting columns (GE HealthCare, Little Chalfont, UK) following the manufacturer instructions. Circular dichroism and tryptophan fluorescence spectra were employed to check the proper folding of the purified enzymes [25 (link)]. The protein concentrations were determined from the absorbance at 280 nm in the presence of 6 M guanidium hydrochloride prepared in 50 mM sodium phosphate at pH 6.5. The extinction coefficients of the wild-type and mutant proteins were calculated as previously described [26 (link); 27 ].
Nickel nitrilotriacetic acid resin
Manufactured by Qiagen
Sourced in United States, Germany
Nickel-nitrilotriacetic acid resin is a chromatography resin used for the purification of histidine-tagged recombinant proteins. It is composed of nickel-loaded nitrilotriacetic acid immobilized on agarose beads. The resin binds to the histidine tag on the target protein, allowing it to be separated from other cellular components.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Escherichia coli bl21 de3, by Agilent Technologies (2 mentions)
Pmal c2x vector, by New England Biolabs (2 mentions)
Hitrap desalting column, by GE Healthcare (1 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
E coli m15, by Qiagen (1 mentions)
Superdex 75, by GE Healthcare (1 mentions)
Chelex 100 resin, by Merck Group (1 mentions)
Nanodrop nd 1000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Phenyl sepharose column, by GE Healthcare (1 mentions)
Ti90 rotor, by Beckman Coulter (1 mentions)
Complete protease inhibitor mixture, by Roche (1 mentions)
Emulsiflex c5, by Avestin (1 mentions)
Lysis buffer, by Qiagen (1 mentions)
Ab5000, by Abcam (1 mentions)
E coli bl21 de3 cells, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)
γ 32p gtp, by PerkinElmer (1 mentions)
Pqe 80l expression vector, by Qiagen (1 mentions)
Mouse arc cdna, by Thermo Fisher Scientific (1 mentions)
35 protocols using nickel nitrilotriacetic acid resin
1
Expression and Purification of Sfβgly Enzymes
2
Construction and Expression of Endosialidase-Fluorescent Fusion Proteins
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The pQE31-based constructs pFEndoNA2 and pFEndoNA for inactive and active endosialidase-GFP fusion proteins, respectively, were available from previous work68 (link). For DsRed fusion derivative, the gfp gene in pFEndoNA2 was replaced with NotI and SpeI restriction sites and a PCR-amplified DsRed-Monomer coding sequence (Clontech). Briefly, linear pFEndoNA2 without the gfp gene was amplified using inverse PCR with Phusion High-Fidelity DNA Polymerase (Thermo Scientific) and 5% DMSO as a PCR additive as recommended by the manufacturer. The purified PCR product was treated with DpnI in order to digest the parental plasmid strands and ligated with the DsRed-Monomer coding sequence, yielding the plasmid pDsEndoNA2. The construct was verified by DNA sequencing at the Institute of Biotechnology at the University of Helsinki.
Constructs were expressed in E. coli M15 (pREP4) expression strain as N-terminal histidine-tagged fusion proteins and purified under native conditions using nickel-nitrilotriacetic acid resin (Qiagen) as described before68 (link).
Constructs were expressed in E. coli M15 (pREP4) expression strain as N-terminal histidine-tagged fusion proteins and purified under native conditions using nickel-nitrilotriacetic acid resin (Qiagen) as described before68 (link).
3
Purification of Recombinant Human PCBP2
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Plasmid pQE-30-PCBP2 (Qiagen) encoding the full-length ORF for human PCBP2 with an in-frame polyHIS tag at its 5′ end was provided by Dr. Ian Brierley (Department of Pathology, University of Cambridge). The plasmid was used to transform E. coli M15 (Qiagen). The transformed cells were grown overnight at 37 °C in LB containing both 35 μg/ml kanamycin and 150 μg/ml ampicillin. Subsequent culturing, IPTG-mediated induction of protein expression, cell lysis, and lysate clarification were carried out as described above for nsp1β. Clarified lysate was mixed end-over-end for 1 h at 4 °C with nickel-nitrilotriacetic acid resin (Qiagen) pre-equilibrated with lysis buffer. The lysate/resin slurry was then poured into a gravity column and washed with 10 column volumes of lysis buffer, followed by 10 column volumes of lysis buffer supplemented with 30 mm imidazole, and finally eluted with lysis buffer supplemented with 250 mm imidazole. The eluted protein was dialyzed against 2 liters of buffer (1× PBS, pH 7.4, 100 mm KCl, 5% glycerol) overnight at 4 °C and then further purified by gel filtration using a Superdex75 (GE Healthcare) gel filtration column. The concentration of purified PCBP2 was quantified using a NanoDrop instrument (A280, ε/1000 = 45,525 m −1 cm−1). PCBP2 mutants (N325D, R40A/N325D, and R40A/R57A/N325D) were purified using the same method as described for the WT protein.
4
Recombinant Silk Protein Expression
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The cDNA encoding TuSp2-RP (90 amino acid residues) with a linker at its N-terminus (52 amino acid residues excluding the first proline residue) was amplified from B6 cDNA using one pair of primers:
5′-CGCGGATCCAACCTGAGCATTGGCGATACC-3′
5′-AATCTCGAGTTAGCCAATTTCCACCATTTTTTT-3′
The PCR product was subcloned into a pET-M expression vector between BamH I and Xho I restriction sites. pET-M was derived from pET-32a (+) expression vector (Novagen) by the removal of Thioredoxin tag, S tag, Nco I and EcoR V restriction sites. The recombinant protein with a hexa-His tag (MHHHHHHSSGLVPRGS) at the N-terminus was expressed in E.coli BL21 (DE3) in LB medium. The cells were grown in 1 L medium with 100 µg/mL of ampicillin at 37 °C. When the OD600 reached ~0.5, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM to induce silk protein expression for 12 hours at 20 °C. The cells were re-suspended in 20 mM Tris buffer (pH 8.0, 300 NaCl and 10 mM imidazole) and sonicated for 30 min at 4 °C. The recombinant protein was purified with nickel-nitrilotriacetic acid resin (Qiagen) under native condition according to the manufacturer′s instructions. Imidazole in the eluted protein solution was removed by dialysis against a PBS buffer at 4 °C. Finally, the purified protein solution was stored at 4 °C without agitation.
5′-CGCGGATCCAACCTGAGCATTGGCGATACC-3′
5′-AATCTCGAGTTAGCCAATTTCCACCATTTTTTT-3′
The PCR product was subcloned into a pET-M expression vector between BamH I and Xho I restriction sites. pET-M was derived from pET-32a (+) expression vector (Novagen) by the removal of Thioredoxin tag, S tag, Nco I and EcoR V restriction sites. The recombinant protein with a hexa-His tag (MHHHHHHSSGLVPRGS) at the N-terminus was expressed in E.coli BL21 (DE3) in LB medium. The cells were grown in 1 L medium with 100 µg/mL of ampicillin at 37 °C. When the OD600 reached ~0.5, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM to induce silk protein expression for 12 hours at 20 °C. The cells were re-suspended in 20 mM Tris buffer (pH 8.0, 300 NaCl and 10 mM imidazole) and sonicated for 30 min at 4 °C. The recombinant protein was purified with nickel-nitrilotriacetic acid resin (Qiagen) under native condition according to the manufacturer′s instructions. Imidazole in the eluted protein solution was removed by dialysis against a PBS buffer at 4 °C. Finally, the purified protein solution was stored at 4 °C without agitation.
5
Purification of Lethocerus Troponin C Domains
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Full-length Lethocerus F2TnC and the two isolated N and C lobes, spanning residues 1–88 and 88–158, respectively, were produced by overexpression in Escherichia coli. The purification procedure was the same as the ones described previously (25 (link)). In short, all constructs were cloned in a pET24d (M11) expression vector (Novagen) containing an N-terminal hexahistidine tag followed by a tobacco etch virus protease cleavage site. The overexpressed proteins were purified by affinity using nickel-nitrilotriacetic acid resin (Qiagen) in two steps separated by an overnight tag cleavage step using in-house produced tobacco etch virus protease. The proteins were then passed through a size exclusion Superdex75 column in 20 mm Tris-HCl buffer at pH 6.8 with 100 mm KCl to remove any high molecular weight contaminants. Proteins were then concentrated on Vivaspin to 0.8 mm samples. Calcium depletion was achieved by a Chelex100 resin (Sigma) and adding 250 mm EDTA to the solution. The holo samples were obtained by adding a 5-fold molar excess of CaCl2.
6
Expression and Purification of Human NAMPT
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cDNA sequence of human NAMPT was amplified by PCR from pGex-6p-3-hNAMPT plasmid (kindly gift from Dr. Shui-Qing Ye in University of Missouri) using the following primers: forward, 5′-GGACATATGATGAATCCTGCGGCAGAAGC-3′; and reverse, 5′-AATCTCGA -GGTAATGATGTGCTGCTTCCAGTTC-3′. The PCR products were digested and cloned into pET21a+ vector using NdeI and XhoI restriction enzyme. The resulting vector was introduced into BL21–CondonPlus (DE3)–RIL. The expression of N-terminal His-tagged NAMPT was induced by 0.5 mM isopropyl-β-D-thiogalactopyranoside at an optical density of 0.6–0.8 at 28 °C for 8 h in 2×YT medium containing 100 μg/ml kanamycin and 37 μg/ml chloramphenicol and then purified with nickel–nitrilotriacetic acid resin (QIAGEN). The purity of the protein was verified more than 90% by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Coomassie staining.
7
RGDV Virion Binding to Sperms
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Mature, live sperms from the seminal vesicles were smeared on poly-lysine-treated glass slides, and then incubated with purified RGDV virions (0.01 μg/μl) for 5, 30, or 90 min For detecting the binding of P2 or P8 of RGDV with sperm in vitro, His-tag-fused P2 or P8 was expressed in Escherichia coli strain Rosetta, and the proteins were purified using nickel-nitrilotriacetic acid resin (Qiagen). Sperm smears were also incubated with the purified proteins (0.5 μg/μl) for 60 min and then stained with P2- or P8-specific IgG conjugated to rhodamine, P2-rhodamine (0.5μg/μl) or P8-rhodamine (0.5 μg/μl), respectively, and then processed for confocal microscopy. Alternatively, purified RGDV virions were pre-incubated with P2 or P8 antibodies (0.5 μg/μl) for 10 min, and then incubated with live sperms. The samples were stained with virus-rhodamine and DAPI, and then processed for confocal microscopy.
8
Purification of His-Tagged Proteins
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The harvested pellet from each induced culture was resuspended in lysis buffer (10% glycerol, 50 mM Tris [pH 8], 100 mM NaCl, 20 mM imidazole) and disrupted at 15,000 lb/in2 on a Microfluidics M-110P homogenizer (Microfluidics, Westwood, MA) at 4°C. The lysate was then centrifuged at 18,000 × g for 30 min at 4°C. The supernatant was applied to a gravity column containing nickel-nitrilotriacetic acid resin (Qiagen), washed with lysis buffer, and eluted in elution buffer (10% glycerol, 50 mM Tris [pH 8], 100 mM NaCl, 250 mM imidazole). The protein samples were dialyzed overnight at 4°C in dialysis buffer (50 mM Tris [pH 8], 100 mM NaCl), and protein concentrations were measured with a NanoDrop ND-1000 UV-Vis spectrophotometer (Thermo Scientific, Waltham, MA); NanoDrop values were corrected on the basis of theoretical extinction coefficients. For the results of SDS-PAGE analysis of all of the purified proteins, see Fig. S4 in the supplemental material.
9
Purification of Phytochrome Holoprotein
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Constructs encoding DrCBDmon variants were transformed into BL21 (DE3) expression cells and grown at 37°C in LB-amp (0.1 mg/ml ampicillin). At OD600 0.5, cells were induced with isopropyl—β-D-1-thiogalactopyranoside at 28°C. Cells were harvested after 4 h by centrifugation at 5000 × g for 30 min, resuspended in lysis buffer (25 mM Tris buffer, pH 8.0, 50 mM NaCl, 5 mM imidazole), and lysed by sonication. After clarification by centrifugation at 40,000 × g for 30 min, the supernatant was incubated with a final concentration of 0.16 mM BV (Frontier Scientific Inc., Logan, UT) in the dark overnight. Proteins were affinity-purified under green light using nickel-nitrilotriacetic acid resin (Qiagen, Valencia, CA). Further purification was performed using hydrophobic interaction on a phenyl-Sepharose column (GE Healthcare) to separate apo- and holophytochrome. Ammonium sulfate was added to the protein at a final concentration of 0.35 M prior to loading. All buffers were filtered and degassed before use. Purified samples were dialyzed overnight against a 200-fold excess volume of (30 mM Tris·HCl, pH 8.0). Finally samples were concentrated to 20 mg/ml, flash-vitrified, and stored at −80°C. Unless otherwise indicated, the samples were kept in the dark before and during the experiments.
10
Purification of Truncated ProOmpA-DHFR Fusion
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The preprotein construct (pOD) consisted of the precursor form of outer membrane protein A, proOmpA, which was truncated at position 69 (11 (link), 12 (link)) and connected at its C terminus to the cytosolic enzyme dihydrofolate reductase. pOD was expressed in MM52 temperature-sensitive cells. 100 ml of 2× YT culture of transformed MM52 cells, supplemented with 100 μg/ml ampicillin, were grown at 30 °C to an optical density of 1 (measured at a wavelength of 600 nm). The culture was then added to 900 ml of prewarmed 2× YT medium, supplemented with 100 μg/ml ampicillin, and grown at 37 °C for another 30 min. Overexpression was induced by the addition of 2 g/liter arabinose, and cells were harvested after 2 h of growth at 37 °C. Cells were lysed in buffer containing 50 mm Tris, pH 7.5, 300 mm KCl, 10% glycerol, 1 mm tris(2-carboxyethyl)phosphine, and cOmplete protease inhibitor mixture (EDTA-free, Roche Applied Science) using an EmulsiFlex C-5 (Avestin) at 20,000 p.s.i. The soluble fraction of the lysate was isolated by ultracentrifugation for 60 min at 40,000 rpm, 4 °C (Beckmann Ti90 rotor). The supernatant was incubated with nickel-nitrilotriacetic acid resin (Qiagen) for 60 min at 4 °C. Bound protein was washed in the presence of 20 mm imidazole, eluted with buffer containing 50 mm Tris, pH 7.5, 100 mm KCl, 10% glycerol, and 300 mm imidazole, and stored at −80 °C.
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