The harvested bacterial pellets were suspended in a binding buffer (50 mM Tris, 150 mM NaCl and 20 mM imidazole) and lysed by freeze-thawing three times with 1 mg/mL lysozyme and ultrasonicator in ice. After centrifugation at 18,000 rpm for 30 min, the supernatant fraction was subject to a prepacked nickel column (Histrap FF crude, Cytiva, USA) and then loaded to a gel filtration column (HiPrep Sepharcryl S-200, Cytiva, USA) after being concentrated by a 30 kDa cut-off ultrafiltration device (50 mL spin column, Millipore, USA) (Fig. S11 ). The protein concentration of CRAB TrxR was determined by measuring FAD absorbance at 463 nm with the extinction coefficient of 11,300 M−1 cm−1 as previously described [29 (link),30 ].
Histrap ff crude
Manufactured by Cytiva
Sourced in Japan
HisTrap FF crude is a prepacked column used for the purification of histidine-tagged proteins. It is designed for rapid and efficient capture of target proteins expressed with a histidine tag. The column contains a precharged Ni Sepharose High Performance medium that selectively binds to histidine-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pet 42a plasmid vector, by Merck Group (2 mentions)
Hiload 26 600 superdex 200, by GE Healthcare (2 mentions)
Hiprep, by Cytiva (1 mentions)
Complete mini edta free, by Roche (1 mentions)
Bl21 codonplus de3 rp competent cells, by Agilent Technologies (1 mentions)
Punk instrument, by Unchained Labs (1 mentions)
Superdex 75, by Cytiva (1 mentions)
Isopropyl β d thiogalactopyranoside iptg, by Merck Group (1 mentions)
In fusion hd cloning, by Takara Bio (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Neb5a, by New England Biolabs (1 mentions)
Pet14b, by GenScript (1 mentions)
T4 polynucleotide kinase, by Promega (1 mentions)
Amicon ultra 0.5 ml centrifugal filter, by Merck Group (1 mentions)
Emulsiflex c3 microfluidizer, by Avestin (1 mentions)
Hitrap q column, by Cytiva (1 mentions)
Hitrap, by Cytiva (1 mentions)
His tagged tev protease, by Merck Group (1 mentions)
10 protocols using histrap ff crude
1
Purification of CRAB TrxR Protein
2
tRNA Demethylation by AlkB Enzymes
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Demethylation reaction was carried out as previously described with modifications (Zheng et al. 2015 (link)). Briefly, truncated wild-type and D135S mutant AlkB with deletion of the amino-terminal 11 amino acids was cloned into a pETBA vector (Biodynamics Laboratory) and overexpressed in Zip Competent Cell BL21 (DE3) (Biodynamics Laboratory). Cells were grown at 37°C in the presence of 50 µM ampicillin until the OD600 reached 0.5–0.6. After the addition of 1 mM IPTG and 5 µM FeSO4, cells were incubated for an additional 4 h at 30°C. Cells were collected, pelleted and resuspended in PBS with protease inhibitor (cOmplete, Mini, EDTA-free, Roche). Then, cells were lysed by sonication and centrifuged at 3000g for 10 min. The soluble proteins were purified using HisTrap FF crude (Cytiva) at 4°C. The reaction buffer contained 300 mM KCl, 2 mM MgCl2, 50 µM (NH4)2Fe(SO4)2·6H2O, 300 µM 2-ketoglutarate, 2 mM l-ascorbic acid, 50 µg/mL BSA, 50 mM MES buffer (pH 5.0). A total of 40 pmol of tRNA was treated with 80 pmol WT AlkB and 160 pmol D135S AlkB in the reaction buffer, and the reaction mixture was incubated at 27°C for 2 h.
3
Recombinant Expression and Purification of cIAP2/XIAP-BIR1 Domains
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The human cIAP2/XIAP-BIR1 domains were cloned in pET28b and expressed and purified, as already described [11 (link)]. Briefly, plasmids for cIAP2-BIR1 or XIAP-BIR1 expression were used to transform Escherichia coli strain BL21-CodonPlus (DE3)-RP competent cells (Agilent). After induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma-Aldrich), bacteria were grown for 3 h at 37 °C. After cell lysis, the expressed proteins were purified using Ni-NTA affinity column (His-trap FFcrude, Cytiva) and size exclusion chromatography (Superdex 75, Cytiva). Proteins were concentrated with Amicon Ultra centrifugal filters (with 3 kDa cut-off) in buffer 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 10 mM DTT. Dynamic Light Scattering assays were performed in pUNK instrument (Unchained Labs) at a concentration of 1 mg/mL and confirmed the high quality of the samples obtained, revealing a sample size polydispersity lower than 20% for all protein constructs and hydrodynamic radii comparable to the monomeric forms of the proteins.
4
Recombinant SARS-CoV-2 Nucleocapsid Protein Purification
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The whole gene of RV-N from a CVS strain was ligated into a pET-42a plasmid vector (Merck, Darmstadt, Germany) using In-Fusion HD Cloning (Takara). The recombinant vector was transformed into Rosetta-gami B (DE3) pLysS Competent Cells (Merck), and the synthesized RV-N was purified with HisTrap FF crude (Cytiva, Tokyo, Japan) under denaturing conditions using 8 M urea.
5
Recombinant RV-N Antigen Production
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Whole RV-N genes derived from the CVS strain were inserted into the pET-42a plasmid
vector (Merck, Germany) using the In-Fusion HD Cloning kit (Takara). The recombinant
vector was transformed into Rosetta-gami B (DE3) pLysS competent cells (Merck), and
recombinant RV-N (rRV-N) was purified using His-trap FF crude (Cytiva, Japan) under
denaturing conditions with 8 M urea. The urea was then removed by dialysis. However, when
the urea concentration decreased, the recombinant protein became insoluble. Therefore, two
recombinant RV-N antigen samples were prepared: an ultimate removal urea and insolubilized
aggregate sample (rRV-N in 0 M Urea) and a solubilized state in a 4 M urea sample (rRV-N
in 4 M Urea). Gene recombination experiments were approved by the Recombinant DNA
Experiment Safety Committee of Kyoto Women’s University (approval number 20-2-03).
vector (Merck, Germany) using the In-Fusion HD Cloning kit (Takara). The recombinant
vector was transformed into Rosetta-gami B (DE3) pLysS competent cells (Merck), and
recombinant RV-N (rRV-N) was purified using His-trap FF crude (Cytiva, Japan) under
denaturing conditions with 8 M urea. The urea was then removed by dialysis. However, when
the urea concentration decreased, the recombinant protein became insoluble. Therefore, two
recombinant RV-N antigen samples were prepared: an ultimate removal urea and insolubilized
aggregate sample (rRV-N in 0 M Urea) and a solubilized state in a 4 M urea sample (rRV-N
in 4 M Urea). Gene recombination experiments were approved by the Recombinant DNA
Experiment Safety Committee of Kyoto Women’s University (approval number 20-2-03).
6
Binding Assay for zfTRF1TB and zfTRF2TB
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EMSA experiments were performed in binding buffer 20 mM Tris pH 8, 150 mM KCl, 0,1 mM EDTA, 1 mM DTT, 500 μg/ml acetylated BSA and 5% glycerol. Proteins and DNA were incubated at room temperature for 15 min and loaded under 50 V on a 5% acrylamide: bisacrylamide (19:1) gel containing 0,5× TBE buffer, after adding 2 μl of a 15% ficoll solution. Migration was performed at 120 V for 2 h at room temperature. Gels were dried and exposed on a phosphorimager screen overnight before analysis using a Phosphorimager Typhoon/FLA 9000. The bottom strand oligonucleotides (show below) were purchased purified from Eurogentec. 5’ labeled using T4 Polynucleotide Kinase (Promega) and hybridized with their corresponding top strand in 20 mM Tris pH 8 50 mM NaCl by heating at 85°C in a 5 l water-bath and slow cooling overnight. Protein expressing vectors pET14b containing the coding sequences of both proteins cloned between the NdeI and BamHI sites were purchased from Genscript. For zfTRF2TB the coding sequence was subcloned into pTrcHisB using the NheI and BamHI sites. Proteins were expressed in NiCo21 (DE3) and NEB5a (New England Biolabs) for zfTRF1TB and zfTRF2TB respectively after induction by 0.1 mM IPTG for 16 h at 25°C. Proteins were purified using a Nickel containing column (HisTrap FF crude, Cytiva) and quantified by Bradford assay.
7
Purification of His-tagged Proteins
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Purification of His-tagged ArcWT and ArcKR was carried out by a third-party company (GenScript Biotech, NJ, USA) using the HD transient expression of recombinant protein—gold system. Briefly, the sequences for ArcWT and ArcKR were synthesized and cloned into the pcDNA3.4 target vector using the EcoRI and HindIII restriction sites. Plasmids were then transfected into the HD 293F cell line for expression. Proteins were purified using HisTrap FF Crude (Cytiva) purification columns and eluted in PBS, pH 7.2. Purity was too low to be detected by SDS-PAGE under non-reducing conditions. After receipt, protein was further concentrated using Amicon Ultra-0.5 ml centrifugal filters (Millipore Sigma) prior to use. Proteins were brought up in the same concentration and used for subsequent SDS-PAGE analysis under reducing [SDS sample buffer containing 5% 2-mercaptoethanol (2-ME)] and nonreducing [SDS sample buffer without (2-ME)] conditions after heating for 5 min at 95°C.
8
Purification of Recombinant C. difficile Toxins
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Plasmid pHis1522 encoding his-tagged TcdB was a kind gift from Hanping Feng and plasmid pHis1522 encoding his-tagged TcdA was a kind gift from Merck. Expression and isolation of recombinant TcdB and TcdA was performed as described by Yang et al. (57 (link)). Briefly, transformed Bacillus megaterium was inoculated into LB containing tetracycline and grown to an A600 of 1.6, followed by overnight xylose induction at 30°C. Bacterial pellets were collected, resuspended with 20 mM Tris pH 8/0.1 M NaCl, and passed twice through an EmulsiFlex C3 microfluidizer (Avestin, Ottawa, ON) at 15,000 lb/in2. The resulting lysate was clarified by centrifuging at 18,000 × g for 20 min. TcdB and TcdA were purified by nickel affinity chromatography followed by anion-exchange chromatography using HisTrap FF Crude and HiTrap Q columns (Cytiva), respectively. Fractions containing TcdB or TcdA were verified by SDS-PAGE, then pooled and diafiltered with a 100,000 MWCO ultrafiltration device (Corning) into 20 mM Tris PH 7.5/150 mM NaCl. Finally, glycerol was added to 5% vol/vol, the protein concentration was estimated by A280, divided into single use aliquots, and stored at −80°C.
9
DARPin Modules Production and Screening
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DARPin modules were raised against HSA, FAP, and CD40 starting from na€ ve DARPin libraries using four to six ribosome display selection rounds (18) . The individual DARPin modules described in the article were screened from the ribosome display pools using highthroughput homogeneous time-resolved fluorescence assays for their binding to HSA, FAP, and CD40, respectively. a-FAPxCD40 and a-mFAPxCD40 were generated by DNA synthesis or standard cloning methods. Single-domain and multidomain DARPin molecules carrying an N-terminal 6xHistidine tag were expressed in Escherichia coli and purified by affinity chromatography followed by a size-exclusion chromatography (Aekta Xpress purification system, His Trap FF Crude from Cytiva and HiLoad 26/600 Superdex 200 from GE Healthcare; ref. 19) .
10
Purification and Complex Formation of Anti-CD40 DARPin
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For crystallization, the N-terminal 6xHis-TEV sequence of the anti-CD40 DARPin domain was removed after purification using a 100-fold excess of His-tagged TEV protease (Sigma, T4455-10KU) in HBSG buffer (20 mmol/L HEPES, 10% glycerol, 300 mmol/L NaCl pH 7.5) with 1 mmol/L DTT and 5 mmol/L EDTA at 4 C (buffer prepared at Molecular Partners using chemicals from Roth AG, Thermo Fisher Scientific AG, and Promega AG). After the addition of 10 mmol/L MgCl 2 , the cleaved DARPin molecule was recovered via negative Ni-NTA followed by SEC (Aekta Xpress purification system using the columns His Trap FF Crude from Cytiva and HiLoad 26/600 Superdex 200 from GE Healthcare) in HBSG. CD40(aa 21-193)-THB-8xHis was expressed in cell culture in the presence of tunicamycin at Proteros Biostructures GmbH. Protein from culture supernatant was purified via Ni-NTA in 50 mmol/L Tris-HCl pH 7.5, 375 mmol/L NaCl, and 10% glycerol and digested with thrombin in 50 mmol/L Tris-HCl pH 7.5, 375 mmol/L NaCl, 20 mmol/L imidazole, and 10% glycerol. The untagged protein was further purified via negative Ni-NTA as above followed by SEC in 10 mmol/L HEPES/NaOH pH 7.0 and 150 mmol/L NaCl. The purified protein was mixed at a 1:1.2 ratio with the anti-CD40 DARPin domain, the complex was purified via SEC equilibrated in 10 mmol/L HEPES/NaOH pH 7.0 and 150 mmol/L NaCl, and concentrated to 36.7 mg/mL.
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