Five mL of overnight pre-cultures were used to inoculate 500 ml of LB supplemented with kanamycin. Cultures were incubated at 37 °C with 180 rpm shaking. When the OD600nm reached 0.6–0.8, the expression of the gene was induced adding 0.1–1 mM IPTG at 18 °C for about 16 hours. Cells were harvested by centrifugation at 12000 rpm for 30 minutes at 4 °C. Cell pellets were resuspended in a lysis buffer (50 mM Tris-HCl pH 7.8, 500 mM NaCl, 10 mM imidazole, 2 mM Beta-mercaptoethanol, 10% (v/v) glycerol) supplemented with EDTA-free protease inhibitor cocktail (Abcam) with a 1:5 ratio (grams of pellet: mL of buffer). Cells were disrupted by sonication and the lysate was clarified by centrifugation for 30 minutes at 4 °C. Poly-histidine-tagged proteins were purified by affinity chromatography using HisGraviTrap (GE Healthcare). The column was equilibrated with 10 ml of lysis buffer before loading the cell lysate and washed with the same buffer containing 50 mM imidazole. Poly-histidine-tagged proteins were eluted with a buffer containing 500 mM imidazole.
His gravitrap
Manufactured by GE Healthcare
Sourced in United Kingdom, United States, Sweden
His GraviTrap is a laboratory equipment designed to separate and concentrate particles from liquid samples. It utilizes gravitational forces to efficiently isolate and collect target analytes or molecules of interest.
Automatically generated - may contain errors
Lab products found in correlation
Shuffle t7 express strain, by New England Biolabs (2 mentions)
Thrombin cleavage capture kit, by Merck Group (2 mentions)
Benzonase nuclease, by Merck Group (2 mentions)
Cellytic b cell lysis reagent, by Merck Group (2 mentions)
Lysozyme, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Bugbuster, by Merck Group (2 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (2 mentions)
Amicon ultra 4, by Merck Group (2 mentions)
Arabinose, by Merck Group (2 mentions)
Catalase, by Merck Group (2 mentions)
Imidazole, by Merck Group (2 mentions)
Chloramphenicol, by Merck Group (2 mentions)
D glucose, by Thermo Fisher Scientific (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (2 mentions)
Enterokinase, by New England Biolabs (1 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
37 protocols using his gravitrap
1
Recombinant Protein Purification from E. coli
2
Nickel Affinity Purification of Proteins
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Sterile filtered yeast supernatant was loaded onto nickel columns (His Gravitrap™, GE Healthcare) and eluted according to the manufacturer’s instructions, using an imidazole buffer (20 mM sodium phosphate pH 7.4, 500 mM NaCl, 500 mM imidazole). Purified protein was immediately re-buffered in sodium phosphate (20 mM, pH 7.4) by means of dialysis (MWCO: 6–8 kDa, Novagen®), after which the protein concentration was spectrophotometrically determined through NanoDrop® (Saveen Werner) and sample purity via a 15% SDS-PAGE.
3
Histidine-tagged Protein Purification
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Cell lysate containing histidine-tagged protein was purified using nickel-affinity-based column systems (His GraviTrap, GE Healthcare) following the manufacturer's protocol. The protein was de-salted by running through a PD-10 desalting column (GE Healthcare). The PD-10 columns were first equilibrated with 25 ml TBS (20 mM Tris/HCl, pH 7.4, and 150 mM NaCl) or PBS (20 mM phosphate, pH 7.4, and 150 mM NaCl). The histidine tag was removed by incubating the protein with enterokinase (New England Biolabs) for 16 h at 23°C. A Superdex 75 16/60 HR column (GE Healthcare) was used for size-exclusion gel chromatography to exclude any impurities from the protein preparation. The protein was concentrated using an Amicon Ultra-15 Centrifugal Filter Unit with Ultracel 3-kDa membrane by centrifugation at 3200 g , 4°C. Protein concentration was determined using a Nanodrop 1000 (Thermo Scientific). An extinction coefficient of 0.863 at λ=280 nm was used for the calculation as determined using Biology workbench [30 (link)].
4
Purification of Mutant TupA Proteins
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After protein expression, the cells were harvested at 5000 × g for 20 min and the pellet was resuspended in a ratio of 2 g cells/mL in 50 mM sodium phosphate buffer (pH 8.0) containing 500 mM NaCl, 5 mM DNase and 1 tablet/L of Protease Inhibitor Cocktail - EDTA (Sigma-Aldrich). The cells were disrupted by sonication and the solution clarified by centrifugation (13000 × g for 30 min). The soluble fraction was filtered through a 0.45 µm membrane.
Protein purification was performed in a one-step protocol using an immobilized-metal affinity chromatography (IMAC), His GraviTrap (GE Healthcare) following the manufacturer’s instructions. Target proteins were eluted using 50 mM sodium phosphate buffer (pH 8.0) containing 500 mM NaCl and 250 mM imidazole. The fractions were analyzed by 10% tris-tricine/polyacrylamide gel electrophoresis stained with Coomassie blue. Fractions containing TupA_R118K, TupA_R118E and TupA_R118Q were dialyzed against 5 mM Tris-HCl pH 7.6. All of the steps were performed at 4 °C.
Protein purification was performed in a one-step protocol using an immobilized-metal affinity chromatography (IMAC), His GraviTrap (GE Healthcare) following the manufacturer’s instructions. Target proteins were eluted using 50 mM sodium phosphate buffer (pH 8.0) containing 500 mM NaCl and 250 mM imidazole. The fractions were analyzed by 10% tris-tricine/polyacrylamide gel electrophoresis stained with Coomassie blue. Fractions containing TupA_R118K, TupA_R118E and TupA_R118Q were dialyzed against 5 mM Tris-HCl pH 7.6. All of the steps were performed at 4 °C.
5
Purification of Recombinant Proteins in E. coli
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Overnight cultures of E. coli BL21 Origami (DE3) containing pET46-Ek/LIC plasmids (Supplementary Table 1 ) were diluted 1:100 and grown to an OD600nm of 0.5 in LB supplemented with 100 μg/ml ampicillin and 1% (w/v) glucose, at 37 °C and 200 rpm. Before induction, glucose from the media was removed and replaced by LB with 100 μg/ml ampicillin. Isopropyl β-D-thiogalactopyranoside (IPTG) was added to a final concentration of: 0.1 mM for rBapB-Spy, 1 mM for rGFP-SpyCatcher and 1 mM for rGFP. The cultures were shaken (150 rpm) at 20 °C overnight. Pellets were resuspended in BugBuster lysis buffer (Novagen) and incubated 30 min RT. Cells were sonicated (3 cycles of 30 s pot 4; 5 cycles of 30 s pot 5) and centrifuged (26,200 × g, 30 min and 4 °C). Supernatants were filtered (0.45 μm). Recombinant proteins were purified by Ni affinity chromatography using HisGraviTrap gravity-flow columns (GE Healthcare) following the manufacturer’s protocol. Detection of recombinant proteins cloned in pET46-Ek/LIC vector was assessed using mouse monoclonal HRP-conjugated anti-polyHistidine antibody (1:2000, Sigma). The concentration of purified proteins was determined using the Bradford Protein Assay (Bio-Rad) with BSA protein as a standard.
6
Recombinant Plasmodium TatD DNase Proteins
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Genes encoding the TatD-like DNase of P. falciparum, P. berghei and P. chabaudi were cloned from the corresponding parasites using the rtPCR method. The sequences coding for the H303 and D352 mutations in PbTatD were chemically synthesized, and all genes were cloned into the pET-28a and pGEX-4T-1 vectors, respectively (Invitrogen). HIS-tagged and GST-tagged recombinant proteins were expressed in BL21(DE3) cells using the pET-28a and pGEX-4T-1 vectors, respectively, and the soluble proteins were purified using His GraviTrap and Glutathione Sepharose (GE Healthcare), respectively, according to the manufacturer's instructions. Anti-rPfTatD/rPbTatD/rPcTatD sera were raised in New Zealand white rabbits by four immunizations with 300 μg of recombinant Protein And Freund's adjuvant. The sera were collected following titre evaluation. Specific IgGs were purified from the sera using Protein A (GE Healthcare) according to the manufacturer's instructions.
7
Purification of His-tagged Proteins
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Chemicals reagents were from Sigma-Merck. Deoxynucleosides, dNMPs, dNTPs, AMP, ADP and ATP were all of >99% purity. Acetic anhydride and H3PO4 (85%) used for the synthesis of acetyl phosphate were ACS grade. Taq DNA polymerase and broad range (10–200 kDa) molecular weight markers were from New England Biolabs (MA, USA). SDS-PAGE gels (15%) were run on a Mini-protein Tetra system and stained with Bio-safe Coomassie Blue (Biorad). His-tagged proteins were purified with His-GraviTrap or His-SpinTrap kits from GE Healthcare.
8
Purification of Recombinant Protein rKAA-1
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E. coli SHuffle T7 Express strain (New England Biolabs), which is an enhanced BL21 derivative engineered to express a chromosomal copy of the disulfide bond isomerase DsbC (Chen et al. 1999 (link)) in the cytoplasm constitutively, was transformed with the rKAA-1 expression vector pET28a-rKAA1 by a conventional method. A transformant was cultured with 1 L of LB media at 37 °C until OD600 reached 0.5, and then IPTG was added at a final concentration of 0.1 mM. After continuously cultured at 20 °C for 16 h, the bacteria cells were harvested by centrifugation at 10,000 g for 20 min. After fracturing the cell by sonication, the His-tagged rKAA-1 (His-rKAA-1) expressed into soluble fraction was purified by His GraviTrap (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instruction. After purification, His-rKAA-1 was dialyzed thoroughly against ultrapure water to remove imidazole which was contained in the elution buffer. Twenty-five milligrams of His-rKAA-1 was then applied to thrombin treatment using a Thrombin Cleavage Capture Kit (Merck) to disconnect the tag peptide from the recombinant according to the manufacturer’s instruction. After removal of thrombin by the above kit, the digested fraction was applied to His GraviTrap to exclude the cleaved His-tag peptide and undigested His-rKAA-1. Generated rKAA-1 was then dialyzed thoroughly against ultrapure water.
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E. coli SHuffle T7 Express strain (New England Biolabs), which is an enhanced BL21 derivative engineered to express a chromosomal copy of the disulfide bond isomerase DsbC (Chen et al. 1999 (link)) in the cytoplasm constitutively, was transformed with the rKAA-1 expression vector pET28a-rKAA1 by a conventional method. A transformant was cultured with 1 L of LB media at 37 °C until OD600 reached 0.5, and then IPTG was added at a final concentration of 0.1 mM. After continuously cultured at 20 °C for 16 h, the bacteria cells were harvested by centrifugation at 10,000 g for 20 min. After fracturing the cell by sonication, the His-tagged rKAA-1 (His-rKAA-1) expressed into soluble fraction was purified by His GraviTrap (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instruction. After purification, His-rKAA-1 was dialyzed thoroughly against ultrapure water to remove imidazole which was contained in the elution buffer. Twenty-five milligrams of His-rKAA-1 was then applied to thrombin treatment using a Thrombin Cleavage Capture Kit (Merck) to disconnect the tag peptide from the recombinant according to the manufacturer’s instruction. After removal of thrombin by the above kit, the digested fraction was applied to His GraviTrap to exclude the cleaved His-tag peptide and undigested His-rKAA-1. Generated rKAA-1 was then dialyzed thoroughly against ultrapure water.
9
Recombinant Rgg1518 Protein Purification
10
Chimeric Fiber Protein: FAdV-4 and FAdV-11
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A chimeric fiber protein retaining epitopes from FAdV-4 and FAdV-11, named crecFib-4/11, was designed and expressed as previously described (16 (link), 17 (link)). Briefly, the open reading frames of fibers from FAdV-4 (fib-2) (reference strain KR5, GenBank accession no. HE608152) and FAdV-11 (field isolate 13/14796) were divided into an amino- and a carboxy-distal segment, and assembled via Gibson assembly cloning. The recombinant protein crecFib-4/11 was expressed in Spodoptera frugiperda Sf9 cells using baculovirus system, and purified via polyhistidine tag on affinity chromatography columns (His GraviTrap, GE Healthcare, Freiburg, Germany) as described by Schachner et al. (8 (link)).
The FAdV-4 field isolate AG234 (GenBank accession no. MK572849) was applied as challenge strain for the animal experiment after being 3-fold plaque purified and propagated on primary chicken-embryo liver cells (20 ). Viral titers were determined by endpoint titration (21 (link)).
The FAdV-4 field isolate AG234 (GenBank accession no. MK572849) was applied as challenge strain for the animal experiment after being 3-fold plaque purified and propagated on primary chicken-embryo liver cells (20 ). Viral titers were determined by endpoint titration (21 (link)).
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