The scFv-SNAP fusion proteins were expressed as previously described [13 (link)]. Briefly, the SNAP-tag and scFv fragment were inserted into the mammalian expression vector pMS. The plasmids were transfected into HEK293T cells using Roti® Fect (Carl Roth, Karlsruhe, Germany). Transfected cells were selected by zeocin (InvivoGen, Toulouse, France) (0.1 mg/mL) supplemented in medium. ScFv-425-SNAP and scFv-EpCAM-SNAP were secreted into culture medium, and then isolated from the supernatant with different imidazole concentrations (10, 40 and 250 mM, respectively) using an Ni-NTA Superflow cartridge (Qiagen, Hilden, Germany) on an ÄKTA start system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden,). All eluted fractions were collected during purification. After incubation with SNAP-Surface® Alexa Fluor® 488 (New Englands Biolabs, Ipswich, MA, USA) at room temperature for 20 min in the dark, fractions were run in 10% SDS-PAGE to confirm the activity of SNAP-tag and the presence of proteins followed by Coomassie brilliant blue staining.
Ni nta superflow cartridge
Manufactured by Qiagen
Sourced in Germany, United States
The Ni-NTA Superflow Cartridge is a laboratory equipment designed for the purification of histidine-tagged proteins. It utilizes a nickel-charged resin to capture and purify the target proteins from complex samples. The cartridge provides a convenient and efficient way to isolate and concentrate histidine-tagged proteins for further analysis or applications.
Automatically generated - may contain errors
Lab products found in correlation
Slide a lyzer, by Thermo Fisher Scientific (5 mentions)
Fluorescence activated cell sorting (facs), by Merck Group (5 mentions)
Sfca filter, by Thermo Fisher Scientific (5 mentions)
Mono s gl, by GE Healthcare (3 mentions)
Sepharose 4b fast flow resin, by Merck Group (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Kta fplc system, by GE Healthcare (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Fast protein liquid chromatography system, by Bio-Rad (2 mentions)
Complete edta free protein inhibitor cocktail, by Roche (2 mentions)
Coomassie blue, by Bio-Rad (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Ppic9k, by Thermo Fisher Scientific (2 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (2 mentions)
E coli bl21 de3, by Takara Bio (2 mentions)
Rotifect, by Carl Roth (1 mentions)
Zeocin, by InvivoGen (1 mentions)
Kta start system, by GE Healthcare (1 mentions)
Up200, by Hielscher (1 mentions)
51 protocols using ni nta superflow cartridge
1
Purification and Labeling of scFv-SNAP Fusion Proteins
2
Expression and Purification of scFv Fusion Proteins
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The scFv-425-SNAP-tag, anti-EpCAM(scFv)-SNAP-tag and anti-CSPG4(scFv)-SNAP-tag fusion proteins were expressed in HEK-293T cells and purified from the supernatant using a Ni-NTA Superflow cartridge (Qiagen, Hilden, Germany) on an ÄKTA FPLC system (GE Healthcare Europe GmbH, Freiburg, Germany) as previously described [12 (link), 13 (link), 40 (link), 41 ].
3
Sortilin Expression and Purification
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Example 3
SORTECDHis was expressed in HEK-293F cells. The His-tag in the proteins enables purification with immobilized metal affinity chromatography. In this process NiNTA Superflow Cartridge (Qiagen) is equilibrated with 50 mM NAH2PO4, 300 mM NaCl and 10 mM Imidazole pH 8.0. Column is loaded with His tagged protein with a residence time of 1 minute. Column is washed with 50 mM NAH2PO4, 300 mM NaCl and 20 mM Imidazole pH 8.0. Protein is eluted with 50 mM NAH2PO4, 300 mM NaCl and 250 mM Imidazole pH 8.0. Subsequently the protein is dialyzed to PBS using a Slide-A-Lyzer with a cut off of 10.000 mwco (Thermo Scientific). After dialyzing the protein is sterile filtered using a 0.2 micron SFCA filter (Thermo Scientific).
The S18-HEK cell line was generated by transfecting HEK293 cells with a human wild type (WT) sortilin expression vector. Stable transfected cells were derived after passage in the presence of a selection agent. Individual clones were selected by dilution cloning. Clones were characterized for sortilin mRNA expression using QPCR. Highest expressing clones were than analyzed by FACS (Guava, Millipore) using an anti-sortilin polyclonal antibody (Polyclonal Goat Sortilin Biotinylated Ab, Cat. No: BAF2934 (R&D Systems)) to determine the surface expressed levels of Sortilin.
4
Sortilin Protein Expression and Purification
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Example 3
SORTECDHis was expressed in HEK-293F cells. The His-tag in the proteins enables purification with immobilized metal affinity chromatography. In this process NiNTA Superflow Cartridge (Qiagen) is equilibrated with 50 mM NAH2PO4, 300 mM NaCl and 10 mM Imidazole pH 8.0. Column is loaded with His tagged protein with a residence time of 1 minute. Column is washed with 50 mM NAH2PO4, 300 mM NaCl and 20 mM Imidazole pH 8.0. Protein is eluted with 50 mM NAH2PO4, 300 mM NaCl and 250 mM Imidazole pH 8.0. Subsequently the protein is dialyzed to PBS using a Slide-A-Lyzer with a cut off of 10.000 mwco (Thermo Scientific). After dialyzing the protein is sterile filtered using a 0.2 micron SFCA filter(Thermo Scientific).
Clones were characterized for sortilin mRNA expression using qPCR. Highest expressing clones were than analyzed by FACS (Guava, Millipore) using an anti-sortilin polyclonal antibody (Polyclonal Goat Sortilin Biotinylated Ab, Cat.No: BAF2934 (R&D Systems)) to determine the surface expressed levels of Sortilin.
5
Purification of Histidine-Tagged Enzymes
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The cell pellets were suspended in 50 ml of equilibration buffer (20 mM phosphate buffer pH 7.5 containing 0.5 M NaCl and 10 mM imidazole) and disrupted by sonication with a Hielscher UP200S ultrasonic processor (Hielscher, Germany) four times for 4 min each. Disrupted cells were centrifuged at 13 000 × g and 4°C for 1 h (Laborzentrifugen, Germany). The crude extract was then collected, filtered and loaded onto a Ni-NTA Superflow Cartridge (Qiagen, Germany) in equilibration buffer. Unbound and weakly bound proteins were washed out using increasing imidazole concentrations. The target enzyme was eluted with purification buffer containing 300 mM of imidazole. The eluted protein was dialyzed three times overnight against 50 mM of phosphate buffer (pH 7.5), after which its purity was checked by SDS–polyacrylamide gel electrophoresis (SDS–PAGE). About, 15% polyacrylamide gels were stained with Instant Blue (Fluka, Switzerland). Protein concentrations were determined by NanoDrop (Sigma-Aldrich, USA). The enzymes were lyophilized using a vacuum pump system for long-term storage.
6
Recombinant Protein Purification and Refolding
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Frozen pellet from induced culture was suspended in cold lysis buffer (10 mM Tris-HCl pH 7.5, 5 mM benzamidine-HCl, 5 mM EDTA, 5 mM DTT, 0.3 mg/ml lysozyme, and 1 mM PMSF) and lysed by sonication on ice. The inclusion bodies were recovered by centrifugation, washed with 50 mM phosphate buffer, 5 mM EDTA, 200 mM NaCl, 0.5 M urea, and 1% Triton X-100, recovered as before, washed with 50 mM phosphate buffer, 1 mM EDTA, and 1 M NaCl, and solubilized in denaturing buffer (10 mM Tris-HCl pH 8.0, 100 mM NaH2PO4, 100 mM NaCl, and 6 M GuHCl). The solubilized inclusion bodies were purified using a Ni-NTA superflow cartridge (Qiagen). Following loading and washing with buffer C (6 M GuHCl, 100 mM NaH2PO4, and 100 mM HCl, pH 6.3), protein was eluted with buffer E (6 M GuHCl, 100 mM NaH2PO4, and 100 mM HCl, pH 4.5). Attempts to dialyze against PBS resulted in protein precipitation; therefore we tested several refolding conditions using a Protein Refolding kit (Pierce, USA). Finally, refolding was done by dilution into refolding buffer RB7 (1 mM GSH, 1 mM GSSH, 1 mM EDTA, 1.1 M GuHCl, 55 mM Tris-HCl, pH 8.0, 21 mM NaCl, and 0.88 mM KCl). The refolded protein was dialyzed against TBS, filter sterilized, and quantitated by BCA method.
7
Optimized Protein Purification Protocol
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These were adapted from the previously described protocols for DM-tRNA-seq5 (link). Briefly, NEB T7 Expression cells were grown in LB media at 37 °C, in the presence of 50 µM kanamycin, to an A600 of 0.6–0.8. Once the cells reached the desired density, IPTG and iron sulfate were added to final concentrations of 1 mM and 5 µM, respectively. After induction, the cells were incubated overnight at 30 °C. Cells were collected, pelleted and then resuspended in lysis buffer (10 mM Tris, pH 7.4, 5% glycerol, 2 mM CaCl2, 10 mM MgCl2, 10 mM 2-mercaptoethanol) plus 300 mM NaCl. The cells were lysed by sonication and then centrifuged at 17,400 rcf for 20 min. The soluble proteins were first purified using a Ni-NTA superflow cartridge (Qiagen) with buffers A (lysis buffer plus 1 M NaCl for washing) and B (lysis buffer plus 1 M NaCl and 500 mM imidazole for elution) and then further purified by ion-exchange (Mono S GL, GE Healthcare) with buffers A (lysis buffer plus 100 mM NaCl) for column loading and B (lysis buffer plus 1.5 M NaCl) for elution.
8
Purification and Characterization of TASKA
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TASKA was purified using a pre-packed, 1-mL nickel-nitrilotriacetic (Ni-NTA) Superflow cartridge (Qiagen, Hilden, Germany) equilibrated with 20 mM sodium phosphate buffer, 500 mM NaCl, and 60 mM imidazole (pH 7.4). The bound enzyme was eluted with a linear gradient of 60–500 mM imidazole at a flow rate of 1.0 mL/min. The active fractions were pooled and dialyzed against 100 mM Tris-HCl buffer (pH 8.0) for 18 h at 4 °C.
The molecular mass and purity of TASKA was determined by 12% (w/v) SDS-PAGE analysis. Zymogram staining for detection of TASKA amylolytic activity was conducted following the method of Yang et al. [67 (link)], except that the 1.0% (w/v) starch solution was prepared in 100 mM Tris-HCl buffer (pH 8.0) and then incubated at 60 °C for 30 min.
The molecular mass and purity of TASKA was determined by 12% (w/v) SDS-PAGE analysis. Zymogram staining for detection of TASKA amylolytic activity was conducted following the method of Yang et al. [67 (link)], except that the 1.0% (w/v) starch solution was prepared in 100 mM Tris-HCl buffer (pH 8.0) and then incubated at 60 °C for 30 min.
9
Cloning and Purification of Molybdenum Cofactor Enzymes
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PfumoaB-WT and EcomogA were cloned into BamHI and HindIII restriction sites of pQE80L (Qiaqen) using pET15b_PfumoaB-WT and pET22b_EcomogA[8] (link) as templates for PCR, resulting in the fusion of an N-terminal His-tag. PfumoaB-H3 was in vitro synthesized by GenScript and cloned into pQE80L in the same way as PfumoaB-WT. E. coli MPT synthase subunits MoaD and MoaE [14] (link), E. coli MoaB, E. coli MogA [8] (link) and A. thaliana Cnx1G [15] (link) were expressed and purified as previously described. For crystallization, PfuMoaB-WT was purified as described [8] (link). For all other procedures PfuMoaB-WT and PfuMoaB-H3 were expressed in E. coli BL21(DE3) for 16 h at 18°C. Expression was induced with 250 µM IPTG at a cell density of OD600 of 0.5. Proteins were purified using ion metal affinity chromatography on Ni2+-nitrilotriacetic (Ni-NTA) matrix (Ni-NTA Superflow Cartridge, Qiagen) attached to Äkta Purifier (GE Healthcare) following the manufacturer’s instructions. Purified proteins were exchanged into buffer containing 0.1 M Tris pH 8.0 and 200 mM NaCl using PD10 columns (GE Healthcare), flash frozen in liquid nitrogen and stored at –80°C until further use.
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PfumoaB-WT and EcomogA were cloned into BamHI and HindIII restriction sites of pQE80L (Qiaqen) using pET15b_PfumoaB-WT and pET22b_EcomogA[8] (link) as templates for PCR, resulting in the fusion of an N-terminal His-tag. PfumoaB-H3 was in vitro synthesized by GenScript and cloned into pQE80L in the same way as PfumoaB-WT. E. coli MPT synthase subunits MoaD and MoaE [14] (link), E. coli MoaB, E. coli MogA [8] (link) and A. thaliana Cnx1G [15] (link) were expressed and purified as previously described. For crystallization, PfuMoaB-WT was purified as described [8] (link). For all other procedures PfuMoaB-WT and PfuMoaB-H3 were expressed in E. coli BL21(DE3) for 16 h at 18°C. Expression was induced with 250 µM IPTG at a cell density of OD600 of 0.5. Proteins were purified using ion metal affinity chromatography on Ni2+-nitrilotriacetic (Ni-NTA) matrix (Ni-NTA Superflow Cartridge, Qiagen) attached to Äkta Purifier (GE Healthcare) following the manufacturer’s instructions. Purified proteins were exchanged into buffer containing 0.1 M Tris pH 8.0 and 200 mM NaCl using PD10 columns (GE Healthcare), flash frozen in liquid nitrogen and stored at –80°C until further use.
10
Recombinant Cyp17a2 Protein Production and Antibody Generation
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The recombinant construct of Cyp17a2 was prepared by cloning the 1566 bp cyp17a2 ORF sequences into a pCold I expression vector. The recombinant plasmid with His-tag at its N-terminal was transformed into Escherichia coli, and expressed with the induction of isopropyl β-D-l-thiogalactopyanoside (IPTG, 1 mmol/L). The His-Cyp17a2 recombinant proteins (25-30 µg) was purified with a Ni-NTA super flow cartridge (Qiagen, Hilden, Germany) and used as an antigen to immunize female rabbits three times at 15-day intervals. Ten days after the last immunization, rabbit serum was collected and purified by affinity chromatography on Sepharose 4B Fast Flow resin (Sigma, Darmstadt, Germany) combined with the Cyp17a2 recombinant protein. To confirm the specificity of the polyclonal antibody, total proteins extracted from testis, ovary, head-kidney, brain, and muscle from 180 dah tilapia were separated using 12.5% SDS-PAGE under reducing conditions. Western blotting was performed according to the methods described previously (29 (link)) and the antibody against tilapia Cyp17a2 was diluted at 1:1000.
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