All proteins were buffer exchanged into EMSA buffer (25 mM HEPES pH 7.2, 200 mM KCl, 1 mM TCEP) using an Illustra NAP-5 column (GE Healthcare). Sample reactions contained 3 µM 6FAM-labelled DNA (listed above) and the indicated amount of protein. The salt concentration was adjusted to 100 mM KCl and 5 mM MgCl2, in a final volume of 20 µl. Reaction mixtures were run for 60 min at 4°C on 0.75% (w/v) agarose gels, at 45 V in 0.5× Tris-borate buffer pH 8.3, in an EM100 gel unit (Cambridge Electrophoresis Ltd) and the gels were visualized under UV light.
Illustra nap 5 column
Manufactured by GE Healthcare
Sourced in United Kingdom
The Illustra NAP-5 columns are a type of lab equipment used for nucleic acid purification. They are designed to efficiently extract and purify DNA or RNA samples from various biological sources. The columns utilize a proprietary resin technology to capture and separate the target nucleic acids, allowing for their subsequent elution and further analysis or processing.
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Lab products found in correlation
Amicon ultra 0.5 ml centrifugal filters, by GE Healthcare (3 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (3 mentions)
Afdye 647 dbco, by Vector Laboratories (2 mentions)
Azide magnetic beads, by Vector Laboratories (2 mentions)
Vivaspin concentrator, by GE Healthcare (2 mentions)
Spectramax m5, by Molecular Devices (2 mentions)
Imagequant las 4010, by GE Healthcare (2 mentions)
Ez link sulfo nhs ss biotin, by Thermo Fisher Scientific (1 mentions)
Tris 2 carboxyethyl phosphine, by Thermo Fisher Scientific (1 mentions)
Femto plasma cleaner, by Diener Electronic (1 mentions)
Aptes, by Merck Group (1 mentions)
Uw2070, by Bandelin (1 mentions)
Econo pac 10dg column, by Bio-Rad (1 mentions)
Protease inhibitor mix hp, by Serva Electrophoresis (1 mentions)
Ni nta spin column, by Qiagen (1 mentions)
Benzonase nuclease, by Merck Group (1 mentions)
Pet200, by Thermo Fisher Scientific (1 mentions)
Bodipy fl gdp, by Thermo Fisher Scientific (1 mentions)
Pd spintrap g 25 column, by GE Healthcare (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
37 protocols using illustra nap 5 column
1
DNA-Protein Binding Assay Protocol
2
Fluorescent Labeling of Vesicles and Transferrin
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Vesicles were labeled for 30 min at room temperature (RT) with a 10 M excess of IRDye800CW-disulfide-N-hydroxysulfosuccinimide (SS-NHS) ester (Li-Cor Biosciences) or a 20 M excess of EZ-Link sulfo-NHS-SS-biotin (Thermo Scientific). Nonreacted linker was quenched by washing with 50 mM Tris (pH 8.0) for 20 min followed by centrifugation at 19,000 × g at 4°C. Vesicles were washed and filtered as described above. Human Tfn (Sigma-Aldrich) was labeled for 2 h at RT with a 6 M excess of IRDye800CW-SS-NHS ester in 0.1 M NaHCO3–PBS using gentle rocking. Nonreacted dye was separated using an Illustra NAP-5 column (GE Healthcare, United Kingdom) and eluted with PBS.
3
Surface Functionalization of nPD Chips
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Chips (1 cm by 1.8 cm) cut from the nPD wafer were surface-treated using a Femto Plasma Cleaner (Diener Electronic) for 5 min. Each chip was immediately submerged in 10.9 ml of methanol, 0.58 ml of Milli-Q water, 0.12 ml of APTES (Sigma-Aldrich), and 0.55 μl of acetic acid and left under agitation overnight. Chips were removed from solution, washed with isopropanol and water three times, dried under nitrogen, and incubated at 90°C for 1 hour. Following the wash and drying steps, each chip was again submerged but, this time, in the solution of 2.2 ml of dichloromethane and 8.2 μl of triethylamine containing 11 mg of 4-maleimidobutyryl chloride. After overnight agitation, they were washed with isopropanol and water three times, dried under nitrogen, and incubated with 3.5 ml of 1× PBS containing 25 μg of GluR2 monoclonal antibody (6C4) (Invitrogen, no. 32-0300) overnight. The antibodies were prepared by adding 50 μl of antibody solution (500 μg/ml) to 450 μl of P/E/MES buffer (5 mM EDTA; Thermo Fisher Scientific) and 10% MES (Fisher Bioreagents) in 1× PBS containing 0.2 μg of tris(2-carboxyethyl)phosphine (Thermo Fisher Scientific), incubating under agitation for 1 hour and exchanging the buffer for 1× PBS using an Illustra NAP-5 column (GE Healthcare).
4
Heterologous Expression of Terpene Synthases
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For heterologous expression, genes were N-terminally truncated (PtTPS19/20: Δ42 aa; PtTPS17: Δ65 aa; PtTPS18: full length, Δ92 aa) and cloned into the bacterial expression vector pET200 (Invitrogen). Cultures of E. coli strain BL21(DE3) were grown at 37 °C and 220 rpm, placed at 18 °C and 180 rpm after reaching an OD600 = 0.5, induced with 1 mM IPTG 60 min later, and grown for another 18 h. The cells were collected by centrifugation (10 min, 5000 g), placed in chilled extraction buffer (50 mM Tris HCl, pH = 7.5, 10 % glycerol (v/v), 10 mM MgCl2, 5 mM dithiothreitol, 5 mM sodium ascorbate, 1× Protease inhibitor Mix HP (SERVA,Germany), 25U Benzonase Nuclease (Merck, Germany), and 0.2 mg/mL lysozyme), and disrupted by a 3 × 30 s treatment with a sonicator (Bandelin UW2070, Berlin, Germany; 50 %). Cell fragments were removed by centrifugation at 14,000 g (10 min, 4 °C) and the supernatant was either directly desalted into assay buffer (10 % glycerol (v/v), 10 mM TrisHCl pH = 7.5, 1 mM dithiothreitol) by passage through an Econopac 10DG column (BioRad, Hercules, CA, USA), or the protein was purified from the supernatant using Ni-NTA Spin Columns (Qiagen, Hilden, Germany) and subsequently desalted through an Illustra NAP-5 Column (GE Healthcare).
5
Fluorescent Labeling and Phosphorylation of eIF2
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For loading of eIF2 with fluorescent nucleotide, 500 μL of eIF2 at 4.9 mg/mL (38 μM) was adjusted to a final concentration of 5 mM EDTA. Bodipy-FL-GDP (Thermo Fisher #G22360) was added to a 5-fold molar excess over eIF2 and the mixture was incubated for 1 hr at room temperature. MgCl2 was added to a final concentration of 10 mM to quench the loading reaction. The mixture was buffer-exchanged by loading onto an Illustra NAP-5 column (GE Healthcare) equilibrated with 20 mM HEPES, 120 mM KCl, 10 mM MgCl2, 1 mM TCEP, pH 7.4. Loaded eIF2 was eluted with 1 mL buffer, aliquoted and stored at −80°C until use.
For phosphorylation of eIF2, 150 μL of eIF2 at 1.5 μM was incubated with 500 μM ATP and 100 nM PERK kinase (Thermo Fisher #PV5106) for 2 hr at room temperature. The mixture was buffer-exchanged by loading onto a PD Spintrap G-25 column (GE Healthcare) equilibrated with 20 mM HEPES, 120 mM KCl, 5 mM MgCl2, 1 mM TCEP, pH 7.4. Phosphorylated eIF2 was eluted with 0.6 mL buffer, aliquoted and stored at −80°C until use.
For phosphorylation of eIF2, 150 μL of eIF2 at 1.5 μM was incubated with 500 μM ATP and 100 nM PERK kinase (Thermo Fisher #PV5106) for 2 hr at room temperature. The mixture was buffer-exchanged by loading onto a PD Spintrap G-25 column (GE Healthcare) equilibrated with 20 mM HEPES, 120 mM KCl, 5 mM MgCl2, 1 mM TCEP, pH 7.4. Phosphorylated eIF2 was eluted with 0.6 mL buffer, aliquoted and stored at −80°C until use.
6
Purification of PA N-terminal Domain
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N-terminal domain of PA subunit (strain A/USA:Huston/AA/1945 H1N1) was delivered by MyBioSource (USA) as a stock solution of 6.3 μM. The His-tag protein was overexpressed in E. coli and purified by nickel affinity chromatography, followed by ion-exchange chromatography, to guarantee the purity higher than 90% (by SDS-PAGE, S1 Fig ). The stock solution was kept in 20 mM Tris-HCl, 0.5 M NaCl, pH 8, 50% glycerol. Prior to experiments, the stock buffer was exchanged using Illustra NAP-5 column (GE Healthcare Life Sciences) according to provided protocol. 250 μl of stock PA-Nter was applied onto column, eluted with 1 ml of buffer (50 mM Hepes and 150 mM KCl pH 7.8) and used further in cleavage studies. Protein-rich fractions contained ~50% of the stock concentration as verified with Micro BCA Protein Assay Kit (ThermoFisher).
7
Recombinant LLO toxin labeling protocol
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Recombinant LLO toxin was from Diatheva and aliquots were stored in 50 mM NaH2PO4, 0.5 M NaCl, 2.7 mM KCl, 1 mM EDTA, 1 mM DTT and 5% (v/v) glycerol at −80°C. FM4-64FX and Fluo-3 were from Molecular Probes, Ionomycin from LC Laboratories and Bapta-AM from Tocris Bioscience. ANXA6-mCherry (pN1-A6-mCherry) was a kind gift from Anette Draeger (University of Bern, Switzerland). To generate the ANXA6-BFP vector, the ANXA6 gene was subcloned into a pTagBFP-N vector using the NheI and XhoI restriction sites. Recombinant LLO toxin was labelled with a tenfold excess of Alexa Fluor 647 with a NHS-ester (Invitrogen) in 50 mM NaH2PO4, 0.5 M NaCl, 2.7 mM KCl, 1 mM EDTA, 100 mM NaHCO3 for 30 min at RT. Unreacted dye was separated on an Illustra Nap-5 column (GE Healthcare Life Sciences) and aliquots were stored in 50 mM NaH2PO4, 0.5 M NaCl, 2.7 mM KCl, 1 mM EDTA, 1 mM DTT and 5% (v/v) glycerol at −80°C. Activity of LLO-A647 was confirmed by using the PI assay to be similar to unlabelled LLO toxin.
8
Preparation of Fluorophore-Labeled DNA Oligonucleotides
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PAGE-purified DNA oligonucleotides with or without fluorophore of FAM or Cy5 were obtained from PURIGO (Taipei, Taiwan). Their sequences are listed in S2 Table . Oligonucleotides were adjusted to 100 μM with 10 mM Tris-HCl pH 8.5. The fluorophore-labeled oligonucleotide was annealed to unlabeled oligonucleotides (S3 Table ) in 10 mM Tris-HCl pH 8.5, 100 mM KCl, and 5 mM MgCl2, with temperatures slowly ramping from 95°C to 25°C at a rate of 0.01°C/sec. Annealed products were separated by polyacrylamide gel electrophoresis in Tris-borate-EDTA buffer (TBE-PAGE). DNA substrates were eluted by immersing gel slices in 0.5 M ammonium acetate at 4°C with rotation overnight. The sample was applied to an Illustra NAP-5 column (GE) and eluted with 10 mM Tris-HCl pH 8.5 with 100 mM NaCl. The concentrations of fluorophore-labeled DNA substrates were measured by TBE-PAGE with fluorophore-labeled DNA oligonucleotides as standards. Design of oligonucleotides is based on a previous reference [58 (link)], with modifications.
9
Antibody Labeling with IRDye800
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Detection antibodies were labeled with IRDye800 though 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide/N-hydroxysuccinimide (EDC-NHS) conjugation. Illustra NAP-5 columns were ordered from GE Healthcare and IRDye 800CW N-hydroxysuccinimide (NHS) ester was purchased from LI-COR Biosciences. IRDye 800CW NHS ester was diluted with dimethyl sulphoxide (DMSO) and detection antibody was mixed at the mole ratio of 1:4 and shaking for 1.5 h in the dark. First, 10 mL of 1 × PBS buffer (pH = 7.4) was added into the column. Then the mixture was added with 1 × PBS buffer (pH = 7.4) to make the total volume of 500 μL after dripping. Finally, IRDye800 labeled detection antibody solution was collected with 500 μL of 1 × PBS buffer (pH = 7.4) and stored at −20 °C in dark before use.
10
Azido-Modified Oligonucleotide Labeling Protocol
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20-nt, 5’-amine-modified readout probes (IDT) were resuspended in 100 mM Sodium Bicarbonate Buffer. Azido-PEG3-SS-NHS (Conju Probe, Cat. #CP-2060) was reacted with 5’ amine-modified oligonucleotides at a 1:100 molar ratio in Sodium Bicarbonate Buffer for at least 6 h or overnight at room temperature on a shaker. Then, the crude mixture was purified using Illustra NAP-5 columns (GE Healthcare, Cat. #17-0853-01) and stored at -20°C. The oligonucleotides were mixed with AFDye 647 DBCO (Click Chemistry Tools, Cat. #1302-1) at a 1:10 molar ratio in Sodium Bicarbonate Buffer for at least 2 h at room temperature and added to Azide Magnetic Beads (Click Chemistry Tools, Cat. #1036-1) at a 1:20 molar ratio for 4 h at room temperature on a shaker. To remove the magnetic beads, the mixtures were placed on a magnet and the supernatant containing dye-labeled cleavable oligonucleotides was removed and stored at -20°C until the seqFISH experiment.
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