The CCN1 coding sequence was cloned in-frame with the glutathione S-transferase (GST) gene at the EcoRI and SalI sites in the pGEX-4T1 vector. Production and purification of the CCN1-GST fusion protein was carried out as described [49 (link)]. Recombinant human estrogen receptor alpha (His-tag) (ab240853) was purchased from Abcam (Cambridge, UK). Ni-NTA His•Bind Resin, a high-performance Ni2+-charged agarose used for rapid one-step purification of proteins containing a His•Tag sequence by metal chelation chromatography, was purchased from Sigma-Aldrich (70666-3). Pierce™ Glutathione Agarose was obtained from ThermoFisher Scientific (San Jose, CA, USA) (16102BID). GST-CCN1 fusion protein was incubated with recombinant human estrogen receptor His-tagged protein and Ni-NTA His beads in the incubation buffer (pH 7.35, 150 mM NaCl, 0.5 mM EDTA, 50 mM Tri-HCl and 0.5% NP-40) at 4° C for 4 hours. After the incubation, the beads were washed for 15 min with washing buffer (pH 7.35, 0.5–1% NP-40+0.5–1% Triton-X100+10% glycerol) on a shaker in the cold room 5 times. The protein complexes bound to the beads were separated with SDS-PAGE and blotted with the indicated antibodies.
Ni nta his bind resin
Manufactured by Merck Group
Sourced in United States, Spain, Germany
Ni-NTA His-Bind Resin is a pre-packed chromatography resin designed for the purification of histidine-tagged recombinant proteins. It utilizes the high affinity interaction between nickel ions (Ni2+) and the histidine tag to selectively capture and purify the target protein.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Bugbuster, by Merck Group (3 mentions)
Mini protean electrophoresis system, by Bio-Rad (3 mentions)
Aminolink plus coupling resin, by Thermo Fisher Scientific (2 mentions)
E coli bl21 star de3 cells, by Thermo Fisher Scientific (2 mentions)
Bca method, by Thermo Fisher Scientific (2 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (2 mentions)
Proteiniso gst resin, by Transgene (2 mentions)
Pierce glutathione agarose, by Thermo Fisher Scientific (1 mentions)
Arabinose, by Thermo Fisher Scientific (1 mentions)
Para azido l phenylalanine, by Bachem (1 mentions)
Snakeskin dialysis tubing, by Thermo Fisher Scientific (1 mentions)
Econo column, by Bio-Rad (1 mentions)
Slide a lyzer mini dialysis device, by Thermo Fisher Scientific (1 mentions)
Pierce detergent compatible bradford assay kit, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 4 centrifugal filter, by Merck Group (1 mentions)
Complete protease inhibitor mixture, by Roche (1 mentions)
Amicon ultra 0.5 centrifugal filter unit, by Merck Group (1 mentions)
Bca assay, by Beyotime (1 mentions)
Clonexpress ultra one step cloning kit, by Vazyme (1 mentions)
52 protocols using ni nta his bind resin
1
GST-CCN1 Protein Interaction Assay
2
Optimizing Protein Expression and Purification
3
Purification of His-tagged PD-L1
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For purification of His‐tagged PD‐L1, a 0.7 × 5 cm Econo‐column (Bio‐Rad) was packed with 1 mL of Ni‐NTA His•Bind® resin (Sigma) and equilibrated with 10 column volumes of 20 mm Na2HPO4, 500 mm NaCl, pH 7.4. The apoplastic fluid was loaded to the column at 1 mL/min and the column was washed with 10 volumes of 20 mm Na2HPO4; 500 mm NaCl, 30 mm imidazole, pH7.4. Bound proteins were eluted with 1 mL of 20 mm Na2HPO4, 500 mm NaCl and 500 mm imidazole (pH 7.4). Fractions containing PD‐L1His were pooled and dialysed overnight against PBS, pH 7.4 using SnakeSkin dialysis tubing (Thermo Fisher Scientific) with a 10 kDa molecular mass cut‐off.
4
Protein-Protein Interaction Assay
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We performed a batch version of the Pull-down assay employed by Padilla-Benavides et al. [7 (link)]. Cell lines stably expressing the dog β1 subunit with an N-terminal 6His-tag (β1His6) were cultured and lysed with RIPA buffer complemented with protease inhibitors; 300 µL of Ni-NTA His•Bind® Resin (Sigma-Aldrich,) previously equilibrated with RIPA buffer, was used to immobilize the β1His6 protein from the lysates. Then, 4 mg of total protein extract was loaded and left to interact for at least 12 h at 4 °C with gentle shaking. After three washes with 500 µL of a 20 mM imidazole solution, 10 mg protein from the different lysates was loaded as prey and left to interact overnight at 4 °C with gentle shaking. After the three washes with 20 mM imidazole, the bound β1His6 was eluted with 200 µL of a 500 mM imidazole solution. Eluates were loaded on a 10% SDS-PAGE gel and analyzed by immunoblotting.
5
Purification of His-Tagged Proteins
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Protein purification using nickel resins (immobilized metal ion affinity chromatography) under native conditions and quantification.
To purify His6 tagged proteins, affinity purification was performed using Ni-NTA His-Bind resin and Ni-NTA buffer kit (Millipore Sigma). Fractions containing the proteins of interest were pooled and were concentrated using Amicon Ultra-4 centrifugal filters, with a 10-kDa cutoff (Millipore Sigma). The concentrated protein fraction was dialyzed against protein storage buffer (50 mM Tris-HCl, 1 mM CaCl 2 , pH 8.0) overnight at 4°C, using a Slide-A-Lyzer mini dialysis device with a 10-kDa molecular-weight cutoff (ThermoFisher Scientific). Protein was quantified using a Pierce detergent-compatible Bradford assay kit, following the manufacturer protocol (ThermoFisher Scientific).
To purify His6 tagged proteins, affinity purification was performed using Ni-NTA His-Bind resin and Ni-NTA buffer kit (Millipore Sigma). Fractions containing the proteins of interest were pooled and were concentrated using Amicon Ultra-4 centrifugal filters, with a 10-kDa cutoff (Millipore Sigma). The concentrated protein fraction was dialyzed against protein storage buffer (50 mM Tris-HCl, 1 mM CaCl 2 , pH 8.0) overnight at 4°C, using a Slide-A-Lyzer mini dialysis device with a 10-kDa molecular-weight cutoff (ThermoFisher Scientific). Protein was quantified using a Pierce detergent-compatible Bradford assay kit, following the manufacturer protocol (ThermoFisher Scientific).
6
In Vitro Protein Kinase Assay
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His6-∆NT-CTR1 and other proteins were expressed in BL21 (Rosetta) and the soluble fraction of total proteins were purified by using Ni-NTA His-Bind Resin (Millipore Sigma, Cat# 70666). A total of 20 ng purified His6-∆NT-CTR1 or His6-∆NT-CTR1ctr1-1 protein was incubated with 100 ng of His6-EIN2-CEND-His6, His6-EBF2, or His6-EIN3 in kinase reaction buffer [50 mM Tris pH 7.5, 10 mM MgCl2, 1× Roche Complete Protease Inhibitor mixture, and 1 µCi [γ−32P] ATP] for 30 min at room temperature. After incubation, the reactions were terminated by boiling in 6× Laemmli SDS sample buffer for 3 min. Samples were subjected to SDS/PAGE, dried, and visualized by autoradiography.
7
Purification of Recombinant DUSP22 Protein
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The BL21(DE3) strain E. coli cells containing pET-28a(+)-DUSP22 were cultured in LB broth and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside at 18 °C overnight. After induction, bacterial cells were centrifuged at 13,000 rpm/4 °C. The bacterial pellet was dissolved in the appropriate volume of lysis buffer (20 mM Tris-HCl (pH 8.0), 0.5 M NaCl and 5 mM imidazole, and 1 mM PMSF), and then lysed by sonication. The lysate was centrifuged at 5000 rpm for 30 min at 4 °C and the supernatant was transferred to the gravity flow column packed with Ni-NTA His-Bind® Resin from Merck Millipore (Burlington, MA, USA). The column was washed (20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 50 mM imidazole) and eluted (20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 300 mM imidazole). The isolated protein buffer was substituted with 30 mM Tris-HCl (pH 8.0) in Amicon Ultra–0.5 Centrifugal Filter Unit (MWCO: 10 kDa) from Merck Millipore. The extracted DUSP22 was stored at −70 °C with 30% glycerol until use.
8
Recombinant Galectin-1 Purification
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Following the protein purification method of Li et al. [23 ], we used the pCold-III-Gal-1 vector (the empty pCold-III vector or the recombinant construct of AcGal-1) constructed by these authors, which was transformed into Escherichia coli BL21 cells. When constructing the plasmid, we added a His-tag to the primer sequence. His-tagged Gal-1 protein expression was induced with 0.1 mM isopropyl-β-d -thiogalactoside and 100 µg/ml ampicillin at 15 °C for 22 h in Luria-Bertani medium. Recombinant protein was purified by Ni-NTA His-Bind-Resin (Merck, Darmstadt, Germany) and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). The BCA assay (Beyotime Institute of Biotechnology, Shanghai, China) was used to measure the protein concentration, following standard protocol.
9
Polyclonal Anti-TCaV1 Antibody Generation
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Polyclonal anti-TCaV1 antibodies were generated in rabbits. The II-III linker of TCaV1 (bases 2188–2550, residues 730–850; GenBank accession number MW915585) was expressed in BL21(DE3) E. coli as a C-terminal 6xHis fusion protein using the expression vector pET-28b(+) (Novagen). Transformed BL21 E. coli were grown to an optical density of 0.5 in 700 ml of Luria Bertani broth. Protein expression was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside for 4 h, then cells were harvested by centrifugation at 4000g for 30 min and sonicated in lysis buffer (500 mM NaCl, 20 mM Tris-HCl 1 mM PMSF, 10% glycerol, 1 mg/ml lysozyme, pH 7.9). Lysed bacteria were centrifuged again at 12,000g for 30 min to separate the supernatant with soluble proteins from the pellet. His-tagged recombinant proteins were purified by Ni2+ affinity chromatography using Ni-NTA His-Bind Resin (EMD Millipore) according to manufacturer instructions, using a 20 mM imidazole column wash solution and 100 mM imidazole elution solution. After elution, the purified proteins were dialyzed with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4). Final yields averaged 0.5 mg/ml. Purified TCaV1 II-III linker peptides were injected into rabbits, and rabbit serum was collected and used for Western blotting and immunostaining experiments. All reagents were obtained from MilliporeSigma.
10
Codon-Optimized Protein Production
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The sequences encoding AJP48854.1, ART39858.1, PHR82761.1, WP_014900537.1, WP_026140314.1, WP_042877612.1, WP_059541090.1, WP_069226497.1, WP_089515094.1 and WP_101198885.1 were used as templates for gene synthesis (GenScript Biotech, EG Rijswijk, The Netherlands), and genes were codon-optimized to maximize expression in Escherichia coli. Genes were flanked by BamHI and HindIII (stop codon) restriction sites and inserted in a pET-45b(+) expression vector with an ampicillin selection marker (GenScript Biotech, Rijswijk, The Netherlands), which was further introduced into E. coli BL21(DE3).
The soluble N-terminal histidine (His) tagged proteins were produced and purified (98% purity, as determined by SDS–PAGE analysis using a Mini PROTEAN electrophoresis system, Bio-Rad, Madrid, Spain) at 4 °C after binding to a Ni-NTA His-Bind resin (Merck Life Science S.L.U., Madrid, Spain), as previously described [4 (link),7 (link)], and stored at −86 °C until use at a concentration of 10 mg mL−1 in 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.0).
The soluble N-terminal histidine (His) tagged proteins were produced and purified (98% purity, as determined by SDS–PAGE analysis using a Mini PROTEAN electrophoresis system, Bio-Rad, Madrid, Spain) at 4 °C after binding to a Ni-NTA His-Bind resin (Merck Life Science S.L.U., Madrid, Spain), as previously described [4 (link),7 (link)], and stored at −86 °C until use at a concentration of 10 mg mL−1 in 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.0).
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