Cells from 50 ml culture were re-suspended in 5 ml of buffer B (8 M urea (Sigma), 0.1 M NaH2PO4 (BDH) and 0.01 M Tris–HCl (Sigma) pH 8.0) and then sonicated in an ice bath for 15 cycles of 10 s with 5 s of cooling between cycles. Lysate was centrifuged at 10 000g for 30–40 min at 4°C to pellet cellular debris, followed by incubation of the supernatant with 20 mM imidazole (Qiagen) and cobalt resin (Fisher Scientific) overnight at 4°C. Supernatant was passed through a gravity column, extensively washed with buffer C (8 M urea, 0.1 M NaH2PO4 and 0.01 M Tris–HCl, pH 6.3) and incubated overnight at 4°C with buffer E (8 M urea, 0.1 M NaH2PO4 and 0.01 M Tris–HCl, pH 4.5). Buffer exchange was performed using PD-10 desalting columns (Amersham Biosciences), replacing the acidic urea buffer with PBS (pH 7.2). Protein concentration was measured using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies) by measuring the absorbance at 280 nm, and proteins were stored at −20°C.
Imidazole
Manufactured by Qiagen
Sourced in Germany
Imidazole is a heterocyclic organic compound that is commonly used in biochemical applications. It serves as a buffer agent and a ligand for affinity chromatography.
Automatically generated - may contain errors
Lab products found in correlation
Imidazole, by Merck Group (3 mentions)
Ni nta agarose bead, by Qiagen (3 mentions)
Kta go system, by Cytiva (2 mentions)
Hitrap desalting column, by Cytiva (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Pd 10 desalting column, by Cytiva (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Complete edta free protease inhibitor, by Merck Group (1 mentions)
Cellfectin 2 reagent, by Thermo Fisher Scientific (1 mentions)
Imidazole, by Cytiva (1 mentions)
Type 50.2 ti rotor, by Beckman Coulter (1 mentions)
Ni nta resin, by Qiagen (1 mentions)
Dnase 1, by Roche (1 mentions)
Vivaspin 20, by Sartorius (1 mentions)
Protease inhibitor cocktail without edta, by Roche (1 mentions)
Anti ha antibody, by Santa Cruz Biotechnology (1 mentions)
8 protocols using imidazole
1
Purification of His-Tagged Proteins
2
Affinity Purification and FKBP1A Ligand Binding Assay
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CHO-DSP1 cells were transfected with control plasmid pcDNA 3.1 (+) or plasmid pcDNA3.1_huFKBP1A-His-Myc [83 (link)]. At 24 hrs post-transfection, cells were lysed in lysis buffer [50 mM NaH2PO4 (Fisher Scientific), 300 mM NaCl (Fisher Scientific), 10 mM imidazole (Sigma Aldrich), 0.05% Tween20 (Sigma Aldrich), pH 8.0] with cOmplete EDTA-free protease inhibitor (Sigma Aldrich) for 30 min on ice. The sample was centrifuged at 3,000 RCF for 10 min at 4°C to remove cell debris, and the supernatant was saved as cell extract. Ten volumes of cell extract were precipitated with 1 volume of pre-equilibrated Ni-NTA agarose beads (Qiagen) at 4°C overnight, followed by washes using wash buffer [50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 0.05% Tween20, pH 8.0]. Meanwhile, MeWo-DSP1 cells were extracted using the above lysis buffer and treated with DMSO, 10 μM of tacrolimus, 10 μM pimecrolimus or 10 μM sirolimus for 30 min at 4°C with end-over-end rotation, followed by incubation with the above-prepared FKBP1A-retaining Ni-NTA agarose beads at 4°C overnight. The bound proteins were eluted from the beads using elution buffer [50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 0.05% Tween20, pH 8.0], followed by western blotting analysis.
3
Purification of SARS-CoV-2 S1-Fc Protein
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After HEK293 cells were transfected and allowed to incubate, they were centrifuged at 2,000 × g for 20 minutes. Recombinant protein constructs all contained signal peptide secretion sequences, and, hence, the supernatant fraction was collected for purification. For affinity purification to the protein’s His-tags, nickel-nitrilotriacetic acid (Ni-NTA) resin (Thermo Scientific #88221) was prepared with two washes for 10 minutes each in 25 mM imidazole (Sigma #I2399) phosphate buffer. Supernatant filtered through 0.22 μm filters was then mixed with the Ni-NTA resin and incubated with agitation overnight at 4°C. Samples were washed three times with 25 mM imidazole phosphate buffer (Sigma #I2399), incubating 5 minutes in between washes. Finally, proteins were eluted using 200 mM imidazole buffer, and resin was separated on polypropylene columns (Qiagen #34924). SARS-CoV-2 S1-Fc protein was purified from serum-free supernatant of transfected 293T cells by Protein A affinity chromatography using hiTrap MabSelect PrismA columns (GE Healthcare) and the Äkta-Pure liquid chromatography system. Experimental replicates all feature different batches of purified protein.
4
Recombinant PfRH5 Protein Purification
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The frozen cell pellet was resuspended in 800 ml of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole (Qiagen); pH 8.0), mixed at 4 °C for 1 h, and lysed by ultrasound technology (UP200S Ultrasonic Processor; 0.7 cycle; 80% Amplitude), followed by centrifugation at 12,000 rpm for 20 min. Supernatant containing protein was applied on 133 µl of nickel Ni–NTA resin (Qiagen) equilibrated with lysis buffer. To remove non-specifically bound proteins, 1 ml of the wash buffer (pH 7) containing 50 mM imidazole was used. Bound proteins were eluted with elution buffer containing 500 mM imidazole. Eluted fractions were analysed by SDS-PAGE and Western blotting. Concentrations of the fractions containing recombinant PfRH5 was determined by Nanodrop (Thermo Scientific) and stored at −80 °C.
5
Recombinant Expression and Purification of CAMSAP2
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Human CAMSAP2 was recombinantly expressed as a fusion protein with N-terminally tagged 6His-GFP in a pFastBac vector (pFastBac+GFP-CAMSAP2 was a gift from Ron Vale (Addgene plasmid # 59037; http://n2t.net/addgene:59037 ; RRID:Addgene_59037) (Hendershott and Vale, 2014 (link))). Baculovirus was produced in Sf21 insect cells after transfection with cellfectin II reagent (Thermo Fisher Scientific). Infected Sf21 cells were incubated at 27 °C for 60 h, followed by harvesting via centrifugation (800 × g for 3 min), flash freezing and storage at –80 °C until protein purification. The insect cell pellet was resuspended in lysis buffer (50 mM HEPES pH 7.6, 300 mM KCl, 1 mM MgCl2, 1 mM EGTA, 1 mM DTT, 0.1% Triton X-100, cOmplete EDTA-free protease inhibitor cocktail (Roche) and PMSF), sonicated for 3 × 1 min with 0.6 amplitude and 0.5 cycles (Hielscher UP50H) and centrifuged for 30 min at 20,000 × g and 4 °C. The supernatant was incubated with Ni-NTA Agarose beads (Qiagen) for 2 h at 4 °C, washed with lysis buffer and eluted with lysis buffer supplemented with 300 mM imidazole (Roth). imidazole was removed via buffer exchange through a HiTrap Desalting column (5 mL, Cytiva) on an ÄKTA go system (Cytiva). Proteins were flash-frozen and stored at −80 °C until usage.
6
Recombinant Ran(Q69L) Protein Purification
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Ran(Q69L), cloned into a pQE32 vector, was transformed into Escherichia coli competent cells (strain BL21-CodonPlus(DE3)-RIL). Expression was induced with 0.2 mM IPTG (Roth) at an optical density of 0.5–0.8 in 2xYT medium. Cells were grown at 30 °C for 16 h. Afterwards, cells were collected, washed with PBS and cell pellet was stored at −80 °C. The pellet was resuspended in lysis buffer (10 mM HEPES pH 7.6, 100 mM KCl, 1 mM MgCl2, 5% v/v glycerol, 1 mM DTT, 0.1% Triton X-100, cOmplete EDTA-free protease inhibitor cocktail (Roche) and PMSF), lysed by sonification and clarified by centrifugation at 40,000 rpm for 20 min in a Type 50.2 Ti Rotor (Beckman Coulter). Supernatant was incubated with Ni-NTA Agarose beads (Qiagen) for 2 h at 4 °C, washed with lysis buffer and eluted with lysis buffer supplemented with 300 mM imidazole (Roth). imidazole was removed via buffer exchange through a HiTrap Desalting column (5 mL, Cytiva) on an ÄKTA go system (Cytiva) operated using the UNICORN software (version 7.6). Ran(Q69L) was concentrated to 350 μM and stored at −80 °C.
7
Purification of Recombinant E. coli BGL Enzymes
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E. coli BL21(DE3) (NEB, Suppl. Table 2 ) transformed with the vectors pET28-bglA or pET28-bglAA781S, were grown at 37 °C under shaking (150 rpm) in 2 x 1 L of LB liquid medium supplemented with 50 μg/mL of kanamycin. 200 μM of isopropyl-thio-β-D-galactoside (Euromedex, France) were added at OD600nm 1 and the growth temperature was switched to 16 °C overnight. Cells were harvested by centrifugation (10 min at 4 °C and 3,000 g) and resuspended in 50 mL of 30 mM Tris-HCl pH 8.0, 5 mM Imidazole, containing few mg of DNAseI (Roche, Mannheim, Germany). Cells were broken using a French Press (Stansted Fluid Power Ltd, Harlow, UK) and the crude extract was subsequently centrifuged (15,000 g, 10 min, 4 °C) to remove cell debris, prior to loading on 3 mL of Ni-NTA resin (Qiagen, Hilden, Germany) equilibrated in 30 mM Tris-HCl, 5 mM Imidazole pH 8.0. The proteins of interest were eluted using 30 mM Tris-HCl, 100 mM Imidazole pH 8.0, buffer. Finally, the purified enzymes were buffer exchanged against 10 mM Tris-HCL pH 8.0 and concentrated by ultrafiltration using Vivaspin20 (cut off 30 kDa, Sartorius, Göttingen, Germany), and stored at −80 °C. The concentration of the proteins was estimated using the absorbance at 280 nm and the absorption coefficient calculated with the program ProtParam tool (www.expasy.org/tools/protparam.html ).
8
Overexpression of SUMO1 in Arabidopsis
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DNA sequences encoding His6 (6 × histidine) and 4 × HA were inserted upstream of Arabidopsis SUMO1 cDNA, and the resulting recombinant His6‐HA4‐SUMO1 DNA was introduced into the β‐estradiol‐inducible vector pER8 61 . The construct was transformed into Arabidopsis by the floral dip method 60 to generate a SUMO1‐overexpressing Arabidopsis line. SUMO1 conjugates were assessed in plants carrying the XVE‐His6‐HA4‐SUMO1 transgene. Plants were grown on MS media for 2 weeks before being treated with 10 μm β‐estradiol for 15 h under light conditions and then being directly exposed to air for 4 h. Samples were harvested, ground in liquid nitrogen, and resuspended in extraction buffer (20 mm Tris/HCl pH 8.0, 8 m urea, 100 mm NaH2PO4, 1% Triton X‐100, 10 mm β‐mercaptoethanol) containing 1× protease inhibitor cocktail without EDTA (Roche, Basel, Basel‐Stad, Switzerland) and 20 mm imidazole (Sigma). After centrifugation, supernatants were purified on Ni2+‐NTA columns using a 20–500 mm imidazole concentration gradient, according to the manufacturer's instructions (Qiagen, Hilden, North Rhine‐Westphalia, Germany). Eluted proteins were detected by western blot analysis with an anti‐HA antibody (Santa Cruz Biotechnology) or an anti‐AtSIZ1 antibody 33 .
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