The hSERT constructs were expressed as C-terminal GFP fusions using baculovirus-mediated transduction of mammalian HEK-293S GnTI− cells, as previously described25 (link),52 (link). Cells were subsequently solubilized in 50 mM Tris pH 8, 150 mM NaCl containing 20 mM DDM, 2.5 mM cholesteryl hemisuccinate (CHS), 0.5 mM dithiothreitol (DTT) in the presence of 1 μM inhibitor (paroxetine, (S)-citalopram, or Br-citalopram). The lysate was passed over 10 ml of Strep Tactin resin, washed with 18 column volumes of 1 mM DDM, 0.2 mM CHS, 5% glycerol, 25 μM lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol at a molar ratio of 1:1:1), and 1 μM ligand in TBS. SERT was eluted in the same buffer containing 5 mM desthiobiotin. The N- and C-terminus containing GFP and purification tags were removed by thrombin digestion and N-linked sugars were truncated using EndoH. SERT was mixed with recombinant 8B6 Fab at a 1:1.2 molar ratio. In the case of Br-citalopram complexed at the central site, Fab purified from hybridoma cells was used. The resulting complexes were further purified by size exclusion chromatography in TBS supplemented with 40 mM n-octyl β-D-maltoside, 0.5 mM CHS, 5% glycerol, 25 μM lipid, and 1 μM inhibitor. The purified SERT-8B6 complex was concentrated to 2 mg/ml−1 and the transporter solution was spiked with 10 μM inhibitor and 1 μM 8B6 Fab, final concentrations, immediately prior to crystallization.
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Desthiobiotin
Desthiobiotin
Desthiobiotin is a chemical compound related to the vitamin biotin.
It is used as a research tool in biochemical and molecular biology applications, particularly in the study of protein-protein interactions and protein purification.
Desthiobiotin can act as a reversibble affinity tag, allowing for the capture and isolation of target proteins.
It has applications in areas such as proteomics, enzyme kinetics, and the development of diagnostic assays.
Researchers utilize desthiobiotin to elevate the reproducibility and accuracy of their experiments, leveraging its unique properties to optimize their Desthiobiotin-related studies.
It is used as a research tool in biochemical and molecular biology applications, particularly in the study of protein-protein interactions and protein purification.
Desthiobiotin can act as a reversibble affinity tag, allowing for the capture and isolation of target proteins.
It has applications in areas such as proteomics, enzyme kinetics, and the development of diagnostic assays.
Researchers utilize desthiobiotin to elevate the reproducibility and accuracy of their experiments, leveraging its unique properties to optimize their Desthiobiotin-related studies.
Most cited protocols related to «Desthiobiotin»
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine
1-palmitoyl-2-oleoylglycero-3-phosphoglycerol
1-palmitoyl-2-oleoylphosphatidylcholine
Baculoviridae
Buffers
Cells
cholesterol-hemisuccinate
Citalopram
Crystallization
desthiobiotin
Digestion
Dithiothreitol
Escitalopram
Glycerin
Hybridomas
Ligands
Lipids
Mammals
Membrane Transport Proteins
Molar
Molecular Sieve Chromatography
Paroxetine
Resins, Plant
Sodium Chloride
Streptococcal Infections
Sugars
Thrombin
Tromethamine
Three micrograms of E. coli RNA was incubated in 50 μl 1× VCE buffer (NEB) supplemented with 0.1 mM S-adenosyl methionine, and 0.5 mM DTB-GTP and 50 units of Vaccinia Capping Enzyme (NEB), for 30 min at 37 °C. The RNA was purified on a Zymo Research Clean and Concentrator-5 column for 200 nucleotide and greater RNA per manufacturer’s instructions with a total of 4 washes with RNA wash buffer. The RNA was eluted in 100 μl of 1 mM Tris pH 7.5, 0.1 mM EDTA (low TE).
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Buffers
calcyclin-associated protein 50
Edetic Acid
Enzymes
Escherichia coli
Nucleotides
S-Adenosylmethionine
Tromethamine
Vaccinia virus
The hSERT constructs were expressed as C-terminal GFP fusions using baculovirus-mediated transduction of mammalian HEK-293S GnTI− cells, as previously described25 (link),52 (link). Cells were subsequently solubilized in 50 mM Tris pH 8, 150 mM NaCl containing 20 mM DDM, 2.5 mM cholesteryl hemisuccinate (CHS), 0.5 mM dithiothreitol (DTT) in the presence of 1 μM inhibitor (paroxetine, (S)-citalopram, or Br-citalopram). The lysate was passed over 10 ml of Strep Tactin resin, washed with 18 column volumes of 1 mM DDM, 0.2 mM CHS, 5% glycerol, 25 μM lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol at a molar ratio of 1:1:1), and 1 μM ligand in TBS. SERT was eluted in the same buffer containing 5 mM desthiobiotin. The N- and C-terminus containing GFP and purification tags were removed by thrombin digestion and N-linked sugars were truncated using EndoH. SERT was mixed with recombinant 8B6 Fab at a 1:1.2 molar ratio. In the case of Br-citalopram complexed at the central site, Fab purified from hybridoma cells was used. The resulting complexes were further purified by size exclusion chromatography in TBS supplemented with 40 mM n-octyl β-D-maltoside, 0.5 mM CHS, 5% glycerol, 25 μM lipid, and 1 μM inhibitor. The purified SERT-8B6 complex was concentrated to 2 mg/ml−1 and the transporter solution was spiked with 10 μM inhibitor and 1 μM 8B6 Fab, final concentrations, immediately prior to crystallization.
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine
1-palmitoyl-2-oleoylglycero-3-phosphoglycerol
1-palmitoyl-2-oleoylphosphatidylcholine
Baculoviridae
Buffers
Cells
cholesterol-hemisuccinate
Citalopram
Crystallization
desthiobiotin
Digestion
Dithiothreitol
Escitalopram
Glycerin
Hybridomas
Ligands
Lipids
Mammals
Membrane Transport Proteins
Molar
Molecular Sieve Chromatography
Paroxetine
Resins, Plant
Sodium Chloride
Streptococcal Infections
Sugars
Thrombin
Tromethamine
E. coli strains BL21(DE3) and BL21(DE3) ΔiscR contained the pACYCDuet-1–hydGX–hydEF plasmid and one of the two pET-21(b) hydrogenase plasmids. Cells were grown in LB Miller growth medium supplemented with kanamycin (40 mg·L−1; only when using the ΔiscR strain), chloramphenicol (25 mg·L−1), ampicillin (100 mg·L−1), 0.5% w/v glucose (∼25 mM), and 100 mM MOPS/NaOH (final pH of medium was 7.4). The ΔiscR strain contains a chromosomal substitution of the iscR gene with another gene conferring resistance to kanamycin [22] (link). 10–50 mL cultures were grown for investigating the effects of cell strains and substrates, while 50–250 mL cultures were grown for hydrogenase purification work. Initially, all cultures were grown aerobically at 25°C until an OD600 of 0.3–0.5. They were then moved into an anaerobic glove box (Coy Laboratory Products) containing 98% N2 and 2% H2 prior to IPTG-based T7 RNA polymerase induction and heterologous protein expression. While ferric ammonium citrate (2 mM) was added to the growth medium prior to inoculation, both cysteine (2 mM) and sodium fumarate (25 mM) were added with IPTG (0.5 mM) within the anaerobic glove box. Cultures were sealed and incubated at 25°C for 16–24 hours following induction.
For investigating media formulations and protein expression by different strains, cells from 1 mL of culture were pelleted at 4,000×g and resuspended in 100 µL of anaerobic BugBuster® Master Mix lysis solution (Novagen) containing an additional 25 mM Tris/HCl (pH 8.0), 25 mM KCl, 3 mM sodium dithionite (DTH), 1 mM dithiothreitol (DTT), 2% v/v glycerol, 0.1% v/v Tween 20, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 2 µM resazurin as an oxygen indicator. After cell lysis (incubation at 25°C for 20 min), lysates were clarified by centrifugation at 14,000×g. Hydrogenase activities in cell lysates were measured using the methyl viologen reduction assay described below. Total protein content of lysates was determined using a commercial assay (Bio-Rad) based on the method of Bradford [42] (link), and the extent of heterologous protein expression was visualized using polyacrylamide gel electrophoresis with SDS-PAGE gels (Invitrogen).
Hydrogenase purifications were carried out while maintaining strict anaerobic conditions. After centrifugation and lysis as described above, approximately 1 mL of Strep-Tactin® Superflow® high capacity resin (IBA GmbH) was used per 50 mL of cell culture for purification. Wash and elution buffers contained the above lysis buffer additives excluding the BugBuster Master Mix and PMSF, and active hydrogenase was eluted with 2.5 mM D-desthiobiotin. Elution fractions were evaluated for active hydrogenase using the methyl viologen reduction assay, and fractions with high activity were pooled. Protein concentrations were measured with the Bradford assay, and hydrogenase iron content was measured using a ferrozine-based colorimetric assay [43] (link). Hydrogenase samples for IR spectroscopic studies were anaerobically concentrated to ∼100 µM using a 10 mL stirred cell and a 5 kD MWCO membrane (Amicon). Hydrogenase samples were not frozen prior to characterization and spectroscopic analysis.
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Most recents protocols related to «Desthiobiotin»
Stable cells were grown in four T75 flasks. Cells were induced with doxycycline at 60–80% confluence. After 18–24 h, the medium in each flask was changed to 7.5 mL of fresh growth medium containing 250 µM desthiobiotin-phenol or biotin-phenol. All DBP and BP parallel experiments were performed simultaneously with the same protocol. The flasks were incubated at 37 °C under 5% CO2 for 30 min according to previously published protocols. Afterward, 750 μL of 10 mM H2O2 (diluted from 30% H2O2, Sigma Aldrich H1009) was added to each flask for a final concentration of 1 mM H2O2, and the flasks were gently agitated for 1 min at room temperature. The reaction was then quenched by adding 7.5 mL of DPBS containing 10 mM Trolox, 20 mM sodium azide, and 20 mM sodium ascorbate to each flask. Then, the solution was removed, and the cells were washed three times with a cold quenching solution (DPBS containing 5 mM Trolox, 10 mM sodium azide, and 10 mM sodium ascorbate). Cells were detached using of cold quenching solution and centrifuged at 1,500×g for 5 min at 4 °C. Cells were resuspended with fresh cold quenching solution and centrifuged again.
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Three sets of samples were prepared for each cell type to investigate the interaction with S. aureus. For the first set, THP-1 cells and HaCaT cells were infected with S. aureus in RPMI 1640 and DMEM media, respectively. The second set comprised THP-1 and HaCaT cells cultured alone in their respective growth media. The third set consisted of S. aureus cultured independently under identical conditions to those of the THP-1 and HaCaT cells (Fig. 1A ). Following infection, cells were scraped off the plates into ice-cold lysis buffer (25 mM Tris HCl, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and 5% glycerol) with protease and phosphatase inhibitors (Pierce Protease and Phosphatase Inhibitor Mini Tablets, EDTA-free, Thermo Fisher). The cell suspension was transferred into 2-mL screw-cap tubes containing 100 µL of 0.1-mm glass beads and lysed with bead-beating. The cell lysates were centrifuged for 20 min at 4°C and 14,000 rpm. The supernatant (total lysate) was transferred to a new tube. The lysis buffer was exchanged for the reaction buffer (4 M urea + lysis buffer) using the Zeba Spin Desalting Columns (Thermo Fisher). Following buffer exchange, the lysate protein concentration was measured using the Qubit protein assay kit, and the protein concentration was adjusted to 2 mg/mL using the reaction buffer. Each sample was mixed with 10 µL of 1 M MgCl2 and incubated for 1 min at room temperature. The ActivX desthiobiotin-ATP probe (Thermo Fisher) was reconstituted in ultrapure water to 20 µM, and 10 µL was added to each sample followed by incubation for 10 min at room temperature. Following labeling, 50 mL of 50% high-capacity streptavidin agarose resin slurry was added to each sample and incubated for 1 hour at room temperature with constant mixing on a rotator. The samples were centrifuged at 100 × g for 1 min to pellet the resin. The resins were washed two times with 500 mL of the reaction buffer, and the bound protein was eluted by adding 2× Laemmli reducing sample buffer and boiled for 5 min.
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HEK293T cells were treated with either ML1-50 (10 µM) or DMSO for 2 h before cell collection and lysis. The proteome concentrations were determined using BCA assay and adjusted to 2 mg/mL. For each biological replicate, 2 aliquots of 1 mL of 2 mg/mL were used (i.e. 4 mg per condition). Each aliquot was treated with 20 µL of IA-alkyne (26.6 mg/mL in DMSO, 200 µM final concentration) for 1 h at RT. Two master mixes of the click reagents were prepared in the meanwhile, each containing 510 µL TBTA (0.9 mg/mL in 4:1 tBuOH/DMSO), 165 µL CuSO4 (12.5 mg/mL in H2O), 165 µL TCEP (14.0 mg/mL in H2O) and 160 µL of either heavy or light isoDTB tags (4 mg in DMSO, Click Chemistry Tools, 1565). The samples were then treated with 120 µL of the heavy (DMSO treated) or light (compound treated) master mix for 1 h at RT. After incubation, one light and one heavy labeled samples were combined and acetone-precipitated overnight at -20 °C. The samples were then centrifuged at 3,500 rpm for 10 min, acetone was removed, and the protein pellets resuspended in cold MeOH by sonication. The samples were centrifuged at 3,500 rpm for 10 min and MeOH was removed (repeated 3× in total). The pellets were dissolved in 600 µL urea (8 M in 0.1 M TEAB) by sonication and the urea concentration was then adjusted to 2 M by adding 1800 µL of TEAB (0.1 M). Two tubes containing solubilized proteins were combined, further diluted with 2400 µL 0.2% NP40 in PBS, and bound to high-capacity streptavidin agarose beads (200 µL/sample, ThermoFisher, 20357) for 1 h at RT with mixing. The beads were then centrifuged for 1 min at 1,000 g, the supernatant was removed, and the beads were washed 3 times with 0.1% NP40 in PBS, 3 times with PBS and 3 times with H2O. The samples were then resuspended in 8 M urea (600 µL in 0.1 M TEAB) and treated with DTT (30 µL, 31 mg/mL in H2O) for 45 min at 37 °C. They were then reacted with iodoacetamide (30 µL, 74 mg/mL in H2O) for 30 min at RT, followed by DTT (30 µL, 31 mg/mL in H2O) for 30 min at RT. The samples were diluted with 1800 µL TEAB (0.1 M), centrifuged for 1 min at 1,000 g, and the supernatant was removed. The beads were resuspended in 400 µL urea (2M in 0.1 M TEAB), and trypsin (8 µL, 0.5 mg/mL) was added and incubated for 20 h at 37 °C. The samples were then diluted with 800 µL 0.1% NP40 in PBS and the beads were washed 3 times with 0.1% NP40 in PBS, 3 times with PBS, and 3 times with H2O. Peptides were then eluted with 0.1% formic acid in 50% acetonitrile (3 × 400 µL). The samples were then dried using a vacuum concentrator at 30 °C, resuspended in 300 µL 0.1% TFA in H2O, and fractionated using high pH reversed-phase peptide fractionation kits (ThermoFisher, 84868) according to the manufacturer's protocol.
Elute from HisTrap consisting of the respective protein fractions were centrifuged at 39,000 g for 15 min, and the supernatant was loaded on a 5 ml StrepTrap HP column (GE HealthCare). The binding buffer was 150 mM NaCl, 50 mM Tris pH 8.0, and the elution buffer was 150 mM NaCl, 50 mM Tris pH 8.0, 2.5 mM desthiobiotin. Elution buffer was injected into the column in two rounds of 5 ml each (Fraction I and II, respectively). The fractions with the protein were pooled, concentrated, and injected into the Superdex-75 column (GE HealthCare) to elute with a final buffer containing 50 mM NaCl, 50 mM Tris pH 8.0 (A50).
In the case of {ABΔCt}, only HisTrap and StrepTrap were performed, followed by washing off desthiobiotin using 50 mM NaCl, 50 mM Tris pH 8.0 (A50) buffer during protein concentration and consequently stored.
In the case of {ABΔCt}, only HisTrap and StrepTrap were performed, followed by washing off desthiobiotin using 50 mM NaCl, 50 mM Tris pH 8.0 (A50) buffer during protein concentration and consequently stored.
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RNA pull-down assay was carried out using Pierce RNA Pull-Down Kit (Pierce). The wide-type or mutated NEAT1 probe (NEAT1-WT or NEAT1-MUT) was labeled with desthiobiotin and conjugated with streptavidin beads. The beads were then incubated with cell lysates, and the enriched proteins were eluted and analyzed by western blot. Anti-sense NEAT1 probe acted as a negative control.
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Top products related to «Desthiobiotin»
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Desthiobiotin is a chemical compound used as a lab equipment product. It is a structural analog of biotin, a vitamin commonly used in biochemical and molecular biology applications. Desthiobiotin can be used for various research purposes, including affinity purification and protein labeling.
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D-desthiobiotin is a biotin analog that can be used in various laboratory applications. It functions as a high-affinity ligand for streptavidin and avidin, and is commonly utilized in techniques such as affinity purification and detection methods.
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The StrepTrap HP column is a chromatography column designed for the purification of recombinant proteins containing a Strep-tag II affinity tag. The column utilizes Streptavidin-coated agarose beads to selectively bind and capture the target protein, allowing for efficient separation and purification.
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The Pierce Magnetic RNA-Protein Pull-Down Kit is a tool for isolating and studying RNA-protein interactions. It utilizes magnetic beads coated with biotinylated RNA baits to capture and pull down associated proteins from cell lysates or extracts.
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The Pierce RNA 3' End Desthiobiotinylation Kit is a tool used to label the 3' end of RNA molecules with desthiobiotin, a biotin analog. This enables the detection, capture, and analysis of the labeled RNA.
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The StrepTrap column is a laboratory equipment designed for the purification and isolation of streptavidin-tagged proteins. It utilizes a matrix that selectively binds to streptavidin-labeled biomolecules, enabling their efficient separation and recovery from complex samples.
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Desthiobiotin is a biochemical compound that functions as a tool in various laboratory techniques. It is structurally similar to biotin, but lacks the sulfur atom. Desthiobiotin can be used in affinity-based purification methods and as a component in assays where its interactions with streptavidin or avidin are of interest.
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Protease inhibitor cocktail is a laboratory reagent used to inhibit the activity of proteases, which are enzymes that break down proteins. It is commonly used in protein extraction and purification procedures to prevent protein degradation.
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Strep-Tactin resin is a chromatographic resin used for the purification of Strep-tagged proteins. It is based on a modified form of the Streptavidin protein, which has a high affinity for the Strep-tag sequence. The resin can be used in various chromatographic techniques, such as affinity chromatography, to capture and purify Strep-tagged proteins from complex mixtures.
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The StrepTrap HP column is a prepacked affinity chromatography column designed for the purification of streptavidin-tagged proteins. It is composed of a rigid, high-flow agarose matrix with immobilized streptavidin, providing high binding capacity and excellent flow properties.
More about "Desthiobiotin"
Desthiobiotin is a versatile chemical compound closely related to the essential vitamin biotin.
It has become a valuable research tool in the fields of biochemistry and molecular biology, particularly for studying protein-protein interactions and facilitating protein purification.
Desthiobiotin's unique properties make it an excellent reversible affinity tag, allowing researchers to capture and isolate target proteins with high specificity.
This has applications in areas such as proteomics, enzyme kinetics, and the development of diagnostic assays.
Researchers often utilize desthiobiotin in conjunction with complementary tools and techniques, such as the StrepTrap HP column, Pierce Magnetic RNA-Protein Pull-Down Kit, and Pierce RNA 3' End Desthiobiotinylation Kit.
These integrate seamlessly with desthiobiotin-based approaches to optimize experimental workflows and enhance the reproducibility and accuracy of their studies.
Beyond desthiobiotin, its closely related analogue D-desthiobiotin also finds use in research applications.
Additionally, Strep-Tactin resin and protease inhibitor cocktails are commonly employed to further refine and protect desthiobiotin-tagged proteins during purification and analysis.
By leveraging the unique properties of desthiobiotin and related tools, researchers can elevate the quality and reliability of their experiments, leading to more robust and meaningful results in their Desthiobiotin-focused studies.
It has become a valuable research tool in the fields of biochemistry and molecular biology, particularly for studying protein-protein interactions and facilitating protein purification.
Desthiobiotin's unique properties make it an excellent reversible affinity tag, allowing researchers to capture and isolate target proteins with high specificity.
This has applications in areas such as proteomics, enzyme kinetics, and the development of diagnostic assays.
Researchers often utilize desthiobiotin in conjunction with complementary tools and techniques, such as the StrepTrap HP column, Pierce Magnetic RNA-Protein Pull-Down Kit, and Pierce RNA 3' End Desthiobiotinylation Kit.
These integrate seamlessly with desthiobiotin-based approaches to optimize experimental workflows and enhance the reproducibility and accuracy of their studies.
Beyond desthiobiotin, its closely related analogue D-desthiobiotin also finds use in research applications.
Additionally, Strep-Tactin resin and protease inhibitor cocktails are commonly employed to further refine and protect desthiobiotin-tagged proteins during purification and analysis.
By leveraging the unique properties of desthiobiotin and related tools, researchers can elevate the quality and reliability of their experiments, leading to more robust and meaningful results in their Desthiobiotin-focused studies.