Prior to imaging, HEK293 MD2-CD14 TLR4 cells were seeded onto polyethylenimine (PEI)-coated glass bottom μ-dishes and left overnight to grow to confluence. 100 µM NTA-FITC (Santa Cruz Biotechnology, Dallas, TX, USA) were incubated with 500 µM NiCl2 for 1 h at RT, and 10 µM dsPRDX5 was then added for 1 h. Unbound probe was then removed using a PD SpinTrap G-25 column (Cytiva), by following manufacturer’s instructions. Cells were imaged using a Zeiss 980 confocal laser scanning microscope (Zeiss GmbH, Oberkochen, Germany). This microscope is equipped with a 405 diode and Helium-Neon lasers, GaAsP detectors, and additional modifications to allow for long term live cell imaging. These modifications include a chamber that maintains cells at 37 °C, with 5% CO2 in air, and a relative humidity of >95%. Excitation and emission settings were determined using YFP positive cells, prior to the addition of PRDX5-NTA-FITC, to reduce crosstalk between the channels. Excitation for FITC and YFP were at 488 and 514 nm respectively, with the key collection bands being between 495–522 nm for FITC and 565–648 nm, with the channels being collected sequentially.
Pd spintrap g 25 column
Manufactured by Cytiva
Sourced in United States, Germany
The PD SpinTrap G-25 column is a size exclusion chromatography column designed for rapid desalting and buffer exchange of small molecules, peptides, and proteins. It is a single-use, pre-packed column that offers a convenient and efficient way to remove unwanted salts, buffers, or other low-molecular-weight compounds from sample solutions.
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Lab products found in correlation
Alexa fluor 647 maleimide dye, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 maleimide dye, by Thermo Fisher Scientific (2 mentions)
Spectramax spectrophotometer, by Molecular Devices (2 mentions)
Discovermp, by Refeyn (1 mentions)
Amicon filter, by Merck Group (1 mentions)
32p atp, by PerkinElmer (1 mentions)
Instantblue coomassie protein stain, by Abcam (1 mentions)
Image lab 5, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Alhydrogel, by InvivoGen (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Dynasore, by Cayman Chemical (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
12 protocols using pd spintrap g 25 column
1
Imaging PRDX5 Protein Interactions
2
Mass Photometry of Protein Complexes
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One hundred microliters of PARLSkd3 was centrifuged at 18,000g, 30 min at 4°C, exchanged to K100 buffer [50 mM tris-HCl (pH 8.0), 100 mM KCl, 10 mM MgCl2, and 1 mM DTT] containing 5 mM of the desired nucleotide over a PD SpinTrap G25 column (Cytiva), and diluted with the same buffer to submicromolar concentrations during measurements. Measurements were carried out on a Refeyen OneMP mass photometer (Refeyn Ltd.) with a 60-s acquisition time. Movies were analyzed using DiscoverMP (Refeyn Ltd.), and the contrast-to-mass conversion was achieved by calibration using the molecular weight standards bovine serum albumin (66 and 132 kDa), Sgt2 (80 kDa), thyroglobulin (330 kDa), and apoferritin (440 kDa). The events recorded from two to three independent measurements were pooled and fitted to Gaussian distributions to extract the mean molecular mass and relative amount of each peak.
3
Labeling and Disaggregation of α-Synuclein Amyloids
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S9C:WT (1:2) αSyn amyloid fibres were incubated with Alexa Fluor 647 maleimide dye (10‐fold molar excess, Thermo Fisher Scientific) and Biotin‐X, SE dye (2.5‐fold molar excess, Thermo Fisher Scientific) for at least 2 h at room temperature in the dark in HKMT buffer. T111C Hsc70 was incubated with Alexa Fluor 488 maleimide dye (10‐fold molar excess, Thermo Fisher Scientific) for at least 2 h at room temperature in the dark in HKMT buffer. Labelled Hsc70 was then buffer exchanged using PD SpinTrap G‐25 column (Cytiva) pre‐equilibrated in HKMD buffer. Labelled amyloid fibres (1 µM) were incubated with labelled Hsc70 (2 µM), DNAJB1 (1 µM) and Apg2 (0.2 µM, if indicated) in disaggregation buffer for 1 h at 30°C in the dark. The T111C‐Alexa Fluor 488 Hsc70 had the same disaggregation activity as unlabelled T111C‐Hsc70 (Fig EV5 ).
4
Purified mNeonGreen Protein Crystallization
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Purified mNeonGreen in 1 × phosphate buffered saline (PBS) was exchanged into 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.0 using a PD Spintrap™ G-25 column (Cytiva). Crystals were grown at 20 °C using the vapour diffusion method from a solution containing 12 mg/ml mNeonGreen mixed with precipitant containing 25% medium molecular weight polyethylene glycol (PEG) smear and 0.1 M HEPES pH 7.5. Diamond shaped crystals appeared after 24 h and grew to final size after a week.
5
Insulin Reduction Assay Using Thioredoxin
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The insulin reduction assay was based on the protocol described in Ref. [27 (link)]. 50 μΜ Trx2 was pre-reduced with 5 mM DTT at 37 °C for 1 h. The DTT was removed using a PD SpinTrap G-25 column (Cytiva) equilibrated in 100 mM KPOi, pH 7.0, 2 mM EDTA. The protein concentration was determined spectrophotometrically. Trx2 was added to wells of a clear 96-well plate containing 130 μΜ recombinant human insulin (Ref 11376497001, Roche Diagnostics) in 100 mM KPOi, pH 7.0, 2 mM EDTA to a final concentration of 1 μΜ or 5 μΜ. An increase in turbidity was measured by following an increase in the absorbance at 650 nm using a SpectraMax spectrophotometer (Molecular Devices) at RT.
6
Hsc70-mediated Amyloid Disaggregation
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S9C:WT (1:2) αSyn amyloid fibres were incubated with Alexa Fluor 647 maleimide dye (10-fold molar excess, Thermo Fisher Scientific) and Biotin-X, SE dye (2.5-fold molar excess, Thermo Fisher Scientific) for at least 2 hours at room temperature in the dark in HKMT buffer. T111C Hsc70 was incubated with Alexa Fluor 488 maleimide dye (10-fold molar excess, Thermo Fisher Scientific) for at least 2 hours at room temperature in the dark in HKMT buffer. Labelled Hsc70 was then buffer exchanged using PD SpinTrap G-25 column (Cytiva) pre-equilibrated in HKMD buffer. Labelled amyloid fibres (1 µM) were incubated with labelled Hsc70 (2 µM), DNAJB1 (1 µM) and Apg2 (0.2 µM, if indicated) in disaggregation buffer for 1 hour at 30°C in the dark.
7
Fluorescent Protein Labeling Protocol
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Fluorescently labeled proteins were prepared by incubating proteins (1 mg/mL in PBS with 0.1 M K2HPO4, pH 9) with 6 fold molar excess of AF647 NHS ester (Invitrogen A20006) for 1.5 hours at room temperature in the dark. Free dye was removed using 10 K Amicon filters (EMD Millipore) and two successive PD SpinTrap G-25 columns (Cytiva). Dye to protein ratios ranged from 1.5 to 2. Fluorescently labeled and unlabeled nanobody–IL-2 fusions were prepared such that each dose contained 1 nmol nanobody–IL-2 and 1.5 nmol dye.
8
Radiolabeling of Oligonucleotides for Biochemical Analyses
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The F-containing oligonucleotides were radiolabeled with 32P-ATP (PerkinElmer) at the 5′ end by T4-polynucleotide kinase (NEB) following a literature method.20 (link) The radiolabeled oligonucleotides were purified using PD Spin-Trap™ G-25 columns (Cytiva). The samples contained a 2 : 3 molar ratio of the radiolabeled oligonucleotide to the non-radiolabeled oligonucleotide.
9
Glutaredoxin Activity Assay by HED
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The HED assay was performed as described in Ref. [26 (link)]. In brief, 50 μΜ Grx1 and Grx2 were pre-reduced with 5 mM DTT at 37 °C for 1 h. The DTT was removed using PD SpinTrap G-25 columns (Cytiva) equilibrated in 100 mM NaPOi, pH 7.0, 1 mM EDTA. The protein concentration was determined spectrophotometrically. 0.7 mM HED (Sigma-Aldrich 380474) was added to a freshly prepared mixture of 0.1 mg/ml BSA, 200 μM NADPH, 1 mM GSH, and 6 μg/ml glutathione reductase from yeast (Sigma-Aldrich G3664) in 100 mM NaPOi, pH 7.0, 1 mM EDTA in a 96-well plate. After 3 min of incubation at RT, Grx1 and Grx2 were added to the wells at a final concentration of 1 μM, and buffer was added to the reference well. The decrease in absorbance at 340 nm was followed in function of time at RT using a SpectraMax spectrophotometer (Molecular Devices).
10
Cysteine Crosslinking Analysis of VirB
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Prior to the crosslinking reactions, VirB or its mutant variants were incubated with 5 mM Tris(2-carboxyethyl)phosphine (TCEP; Sigma, USA) for 1 h at room temperature to fully reduce all cysteine residues. Subsequently, the protein was transferred to reaction buffer (25 mM HEPES/NaOH pH 7.5, 500 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA) using PD SpinTrap G-25 columns (Cytiva, Germany). Crosslinking was performed at room temperature in mixtures containing 10 µM protein, which were supplemented with 1 mM CTP and/or 1 µM of a virS-containing double-stranded DNA oligonucleotide (virS-icsB-for/virS-icsB-rev) when appropriate. After the indicated incubation times, bismaleimidoethane (BMOE; dissolved in dimethylsulfoxide) was added to a concentration of 1 mM. After brief mixing and incubation for 1 min, the reaction was stopped by the addition of dithiothreitol-containing SDS sample buffer. As a negative control, samples were treated with dimethylsulfoxide instead of BMOE. The samples were then analyzed by SDS-polyacrylamide gel electrophoresis, and protein was stained with InstantBlue Coomassie Protein Stain (Expedeon, Germany). The gels were imaged with a ChemiDoc MP imaging system (Bio-Rad Laboratories, USA), and the intensities of the different bands were quantified using Image Lab 5.0 software (Bio-Rad Laboratories, USA).
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