pRSF-duet-P18NP was transformed into BL21 competent bacterial cells. After overnight IPTG induction (0.1 mM) at 16°C, the cells were lysed and the NP protein was purified on a HisTrap immobilized metal affinity chromatography (IMAC) column (Cytiva, USA). The Nunc MaxiSorp plates (Thermo-fisher, USA) were coated with 200 ng of recombinant NP proteins overnight at 4°C in coating buffer (50 mM Carbonate-Bicarbonate buffer, pH 9.5). Plates were washed once with wash buffer (PBS + 0.2% Tween20) and blocked for 2 h at ambient temperature with blocking buffer (PBS + 4.0% dry milk) followed by three more washes. Serum samples were serially diluted in diluting buffer (PBS + 0.05% Tween20), added to plates, and incubated for 1 h. Following three washes, samples were incubated with goat anti-guinea pig IgG HRP secondary antibody (Sigma, USA) at 1:10,000 dilution for 1 h. Following four washes, samples were incubated with the TMB substrate (Thermo-fisher, USA) for 15 min in the dark, then a stop solution (0.16M H2SO4) was added and optical density (OD) values were read at 450 nm using a Biotek Synergy 2 plate reader (Biotek, USA). The IgG antibody endpoint titer was defined as the highest dilution giving OD450 above the cutoff value, which was determined by average plus four times standard deviation of secondary-antibody-only controls.
Histrap
Manufactured by Cytiva
Sourced in United States
HisTrap is a pre-packed affinity chromatography column designed for the purification of histidine-tagged recombinant proteins. The column contains Ni Sepharose resin, which selectively binds to the histidine tag on the target protein, allowing for its separation from other components in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Biotek synergy 2 plate reader, by Agilent Technologies (1 mentions)
Nunc maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Tmb substrate, by Thermo Fisher Scientific (1 mentions)
Glutamate dehydrogenase, by Merck Group (1 mentions)
Potassium phosphate, by Merck Group (1 mentions)
Acetaldehyde, by Merck Group (1 mentions)
Acetaldehyde dehydrogenase, by Merck Group (1 mentions)
Imidazole, by Merck Group (1 mentions)
L glutamate, by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Vivaspin 20 spin filters, by Cytiva (1 mentions)
Mgcl2, by Merck Group (1 mentions)
Hitrap q column, by Cytiva (1 mentions)
Hepes, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Lysozyme, by Merck Group (1 mentions)
12 protocols using histrap
1
SARS-CoV-2 NP Protein Antibody ELISA
2
Purification and Analysis of Enzymes
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Lysogeny broth (LB) medium, isopropyl-β-d -thiogalactopyranoside
(IPTG), and NADPH were purchased from
Research Products International. The protease inhibitor cocktail,
lysozyme, DNase I, glutamate dehydrogenase, acetaldehyde dehydrogenase,
acetaldehyde, kanamycin, imidazole, Tris, HEPES, PLP, andl -glutamate were obtained from Sigma-Aldrich. Ammonium bicarbonate,
potassium phosphate, 2-mercaptoethanol, KCl, and MgCl2 were
also acquired from Sigma-Aldrich. DNase I was purchased from Roche.
Vivaspin 20 spin filters and HisTrap and HiTrap Q columns were obtained
from Cytiva. The 10 kDa Nanosep spin filters were purchased from Pall
Corporation (Port Washington, NY). Deuterium oxide was acquired from
Cambridge Isotope Laboratories Inc., and oxygen-18 labeled water (98%)
was obtained from Medical Isotopes Inc.
(IPTG), and NADPH were purchased from
Research Products International. The protease inhibitor cocktail,
lysozyme, DNase I, glutamate dehydrogenase, acetaldehyde dehydrogenase,
acetaldehyde, kanamycin, imidazole, Tris, HEPES, PLP, and
potassium phosphate, 2-mercaptoethanol, KCl, and MgCl2 were
also acquired from Sigma-Aldrich. DNase I was purchased from Roche.
Vivaspin 20 spin filters and HisTrap and HiTrap Q columns were obtained
from Cytiva. The 10 kDa Nanosep spin filters were purchased from Pall
Corporation (Port Washington, NY). Deuterium oxide was acquired from
Cambridge Isotope Laboratories Inc., and oxygen-18 labeled water (98%)
was obtained from Medical Isotopes Inc.
3
Purification of Oligomeric p62 Mutant
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The oligomerization deficient p62 truncation p62(1–122, D69A/D71A/D73A) was cloned into pCoofy4 (Ref: PMID: 23410102) using restriction-free cloning (van den Ent and Löwe 2006 (link)), transformed into the Rosetta 2(DE3) strain, and expressed in M9 medium with 15NH4Cl as the sole nitrogen source at 22°C O/N upon induction with 0.5 mM IPTG. The cells were lysed in a buffer containing 50 mM Tris pH 8, 750 mM NaCl, 10% glycerol, 20 mM imidazole, and cOmplete, EDTA-free Protease Inhibitor Cocktail using a microfluidizer (M-110L, Microfluidics Inc.). Subsequently, the protein was purified from the cleared lysate by Nickel affinity chromatography (HisTrap, Cytiva) in the same buffer. The affinity tag was removed by 3C protease cleavage (EMBL, PepCore), dialysis, and an additional passage over the HisTrap column. As a final purification step, oligomers were removed by gel filtration on a Superdex S75 10/300 in a buffer containing 20 mM MES pH 6.5, 100 mM NaCl, 0.2 mM TCEP, and 0.05% NaN3.
4
Purification of Recombinant Human EB2 Protein
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A codon optimized EB2 gene was synthesized by an external company and cloned into the pET40-b with NdeI/BamHI double digestion. The resulting gene encodes human EB2 with an N-terminal 6xHis tag (see the Additional file 1 : Information for the nucleotide sequence). After recombinant expression, E. coli BL21(DE3) recombinant cells were lysed by sonication in 50 mM TrisHCl pH 8.0, 0.5 M NaCl, 5% glycerol, 20 mM imidazole supplemented with a protease inhibitor cocktail. The soluble fraction was recovered after a centrifugation (14,000 g for 45 min at 4 °C) and loaded onto a HisTrap of 1 mL (Cytiva). The target protein was eluted with 250 mM imidazole and loaded onto a a Hiload 16/600 Superdex 200 pg using 50 mM TrisHCl pH 8.0, 0.18 M NaCl as a running buffer for a final polishing step. The final preparation was stored at – 80 °C in 40 mM TrisHCl pH 8.0, 0.15 M NaCl, 1 mM DTT, 15% glycerol at 1.8 mg/mL protein concentration.
5
Purification of Recombinant KaiC Proteins
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KaiC-AA and KaiC-EE were expressed and purified as described previously38 (link). Briefly, KaiC mutants were expressed recombinantly in E. coli with N-terminal His6 tags. Clarified lysate was purified using Ni affinity chromatography (Histrap, Cytiva), fractions containing KaiC were pooled, and the expression tags were cleaved overnight. The resulting material was further purified using size exclusion chromatography (HiPrep S300, Cytiva) and fractions corresponding to the molecular weight of KaiC hexamers were selected.
6
Codon-optimized Fusion Protein Expression and Purification
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MT3 (human)-Omp19 (MO), MT1 (human)-Omp19 (M1O), MT3 (murine)-Omp19 (MmO), MT3 (human)-Hc (MH) and MT3 (human) genes were codon-optimized for expression and synthesized. The proteins were expressed from a pET28a plasmid in E. coli BL21. The fusion proteins were purified from bacterial lysate supernatants by a HisTrap™ affinity chromatography column (Cytiva, Uppsala, Sweden) and DEAE Sepharose™ Fast Flow column (Cytiva, Uppsala, Sweden). The effluents corresponding to the fusion proteins were treated with an endotoxin removing gel (Cytiva, Uppsala, Sweden). The fusion proteins (MO, M1O, MmO, MH, and MT3) were collected, and assessed with SDS-PAGE and Western blot analysis. In Western blot analysis, the bands were probed with Omp19 immunized mice serum and goat anti-mouse IgG Fc (HRP) (Abcam, ab97265, Cambridge, UK) against Omp19, MO, M1O and MmO; with human monoclonal antibody TT0067 and goat anti-human IgG Fc (HRP) (Abcam, ab97225, Cambridge, UK) against Hc and MH, respectively. Hc and Omp19 were prepared as described previously (18 (link), 19 (link)). Lipopolysaccharide (LPS) contamination was identified by the tachyplens amebocyte lysate (TAL) assay (Chinese Horseshoe Crab Reagent Manufactory, Xiamen, China).
7
Recombinant Protein Expression and Purification
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Recombinant proteins were produced in E. coli BL21 (DE3) cells cultured in 500 mL of LB broth containing ampicillin (50 μg·mL−1) at 37 °C (200 rpm). Cells were grown to mid-exponential phase (OD600 ~ 0.4 to 0.6). Overexpression was induced by adding isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and the cultures were further grown at 16 °C (200 rpm) for 18 h. The cells were harvested by centrifugation (7000× g for 20 min at 4 °C), sonicated and His6-tagged recombinant proteins were purified via immobilized nickel affinity chromatography (His-Trap; Cytiva, Marlborough, MA, USA) utilizing a one-step gradient elution up to 100% elution buffer containing 20 mM sodium phosphate, pH 7.4, 500 mM NaCl and 500 mM imidazole using an Akta Pure protein purification FPLC system (Cytiva, Marlborough, MA, USA). The purity of the recombinant proteins was determined by SDS/PAGE and the final concentration was determined from calculated molar extinction coefficients at 280 nm using a Nanodrop One (Thermofisher Scientific, Illkirch, France).
8
Cloning and Protein Expression of AgmT Variants
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DNA sequences encoding amino acids 25 – 339 of AgmT and AgmTEAEA were amplified by polymerase chain reaction (PCR) and inserted into the pET28a vector (Novogen) between the restriction sites of EcoRI and HindIII. The resulting plasmids were transformed into E. coli strain BL21(DE3). Transformed cells were cultured in 20 ml LB (Luria-Bertani) broth at 37 °C overnight and used to inoculate 1 L LB medium supplemented with 1.0% glucose. Protein expression was induced by 0.1 mM IPTG (isopropyl-h-d-thiogalactopyranoside) when the culture reached an OD600 of 0.8. Cultivation was continued at 16 °C for 10 h before the cells were harvested by centrifugation at 6,000 × g for 20 min. Proteins were purified using the NGC™ Chromatography System (BIO-RAD) and a 5-ml HisTrap™ (Cytiva)17 (link),53 (link). Purified proteins were concentrated using Amicon™ Ultra centrifugal filter units (Millipore Sigma) with a molecular cutoff of 10 kDa and stored at −80 °C.
9
Purification of S16 Protein from Bacterial Expression
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For the purification of S16, the
pellet from 2 L expression culture was resuspended in 200 mL of buffer
A2 (20 mM Tris–HCl, 500 mM NaCl, pH 7.5),
supplemented with 8M urea, and incubated with magnetic agitation
at room temperature overnight. After adjusting the pH to 4.0 with
acetic acid, the sample was further incubated for 2 h at room temperature.
Following centrifugation (27,220g, 4 °C, and
30 min), the supernatant was precipitated by adding (NH4)2SO4 to a final concentration of 1.32M and incubated with agitation for another 6 h. The mixture
was centrifuged again, and the resulting supernatant was further precipitated
with 2.80M (NH4)2SO4. The
precipitate was solubilized in buffer A2 containing 8 M urea and loaded
into a 5 mL HisTrap (Cytiva) column preequilibrated with 10 column
volumes (CV) of buffer A2. After washing the column with 10 CV buffer
A2, the sample was eluted using a gradient up to 300 mM imidazole
over 5 CV of buffer B2 (20 mM Tris–HCl, 500 mM NaCl, 300 mM imidazole, pH 7.5). The sample was then dialyzed
against water overnight at 4 °C. During dialysis, S16 remained
in solution, while impurities precipitated out. The protein was concentrated
and then stored at −80 °C for further use. The expression
yield was around 23 mg.
pellet from 2 L expression culture was resuspended in 200 mL of buffer
A2 (20 m
supplemented with 8
at room temperature overnight. After adjusting the pH to 4.0 with
acetic acid, the sample was further incubated for 2 h at room temperature.
Following centrifugation (27,220g, 4 °C, and
30 min), the supernatant was precipitated by adding (NH4)2SO4 to a final concentration of 1.32
was centrifuged again, and the resulting supernatant was further precipitated
with 2.80
precipitate was solubilized in buffer A2 containing 8 M urea and loaded
into a 5 mL HisTrap (Cytiva) column preequilibrated with 10 column
volumes (CV) of buffer A2. After washing the column with 10 CV buffer
A2, the sample was eluted using a gradient up to 300 m
over 5 CV of buffer B2 (20 m
against water overnight at 4 °C. During dialysis, S16 remained
in solution, while impurities precipitated out. The protein was concentrated
and then stored at −80 °C for further use. The expression
yield was around 23 mg.
10
Recombinant Expression and Purification of CDKL5 Catalytic Domain
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To produce the catalytic domain of CDKL5 in E. coli, the primers named PhSumoCDKL5_NdeI_fw and PhCDKL5dC_XhoI_rv were used in a PCR on pB40-BCD2-107 (L). This gene encoding a Sumo-tagged version of CDKL5(1–352) was cloned into the pET40-b vector in frame with a C-terminal 8xHis tag using NdeI/XhoI double digestion. After recombinant expression in E. coli BL21(DE3), the recombinant cells were resuspended in 50 mM TrisHCl pH 8.0, 0.5 M NaCl, 20 mM imidazole and lysed by sonication in the presence of a protease inhibitor cocktail. After centrifugation (14,000 g, 4 °C, 60 min), the soluble fraction was loaded onto a HisTrap of 1 mL (Cytiva) and both the full-length protein, and its N-terminally truncated fragment were collected with a linear gradient of imidazole. A sample containing approximately 30 µg of the intact catalytic domain and 6 µg of the N-terminally truncated fragment were loaded onto SDS-PAGE and then electroblotted onto a PVDF membrane using 10 mM CAPS, 10% methanol pH 11.0 as the transfer buffer. The protein bands were made visible by Ponceau S staining and submitted to Edman sequencing at the Institute of Biosciences and Bioresources (CNR, Naples).
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