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Amicon ultra 0.5 10 k

Manufactured by Merck Group
Sourced in Germany

The Amicon Ultra-0.5 10 k is a centrifugal filter device designed for the concentration and purification of macromolecules. It utilizes a regenerated cellulose membrane with a molecular weight cut-off of 10 kDa to facilitate the separation and recovery of target molecules from complex samples.

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5 protocols using amicon ultra 0.5 10 k

1

Fluorescently Labeled Pol II Phospho-Specific Fabs

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Fluorescently labeled antibody fragments (Fabs) specific to Pol II Ser5Phos and Pol II Ser2Phos were prepared from monoclonal antibodies specific to Pol II Ser5 and Ser2 phosphorylation40 (link),44 (link),77 (link). Monoclonal antibodies were digested with Ficin (ThermoFisher Scientific), and Fabs were purified through protein A-Sepharose columns (GE Healthcare) to remove Fc and undigested IgG. After passing through desalting columns (PD MiniTrap G25; GE Healthcare) to substitute the buffer with PBS, Fabs were concentrated up to >1 mg/ml using 10 k cut-off filters (Amicon Ultra-0.5 10 k; Merck), Fabs were conjugated with Alexa Fluor 488 (Sulfodichlorophenol Ester; ThermoFisher Scientific) or Cy3 (N-hydroxysuccinimide ester monoreactive dye; GE Healthcare) to yield ~1:1 dye:protein ratio. After the buffer substitution with PBS, the concentration was adjusted to ~1 mg/ml.
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2

Optimized Expression and Purification of FP Variants

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The EYFP mutants were generated using IVA-cloning PCR technique [26] (link) with the following oligonucleotide set containing the appropriate substitutions (primer sequences are shown in Suppl. methods). pQE30 plasmid vector backbone (Qiagen) and E. coli XL1 Blue strain (Evrogen) were used for the DNA constructs assembly.
FP variants, cloned into the pQE30 vector (Qiagen) with a 6His tag at the N-terminus, were expressed in E. coli XL1 Blue strain (Evrogen). The proteins were isolated by ultrasonic cell lysis, purified using TALON metal-affinity resin (Clontech), eluted from the adsorbing material by treatment with 100 mM imidazole (pH 8) and then desalted by ultrafiltration with Amicon® Ultra-0.5 10 K (Merck) filters. All isolation and chromatography procedures were performed in PBS buffer pH 7.4 (Gibco). The same solution was further used for the specimen storage and spectroscopy measurements.
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3

Exosome Characterization and Labeling for DEP

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Commercially available exosomes, bovine milk exosome (mean diameter: 119 nm, EXBM100L, COSMO Bio, Co., Ltd., Tokyo, Japan), human breast milk exosome (mean diameter: 174 nm, EXHM100L, COSMO Bio, Co., Ltd., Tokyo, Japan), and human breast cancer (MCF-7) exosome (peak diameter: 100 nm, EXOP-100A-1, System Biosciences, LLC, Palo Alto, CA, USA), were used. The exosomes were fluorescently labeled using ExoSparkler Exosome Membrane Labeling Kit-Green or Deep Red (Dojindo Laboratories, Kumamoto, Japan). Before the DEP experiments, the exosome suspensions were desalted using Amicon Ultra 0.5, 10 k (Merck KGaA, Darmstadt, Germany). The conductivity of the suspension medium was adjusted using NaCl solution.
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4

Antibody-MtsA Binding Kinetics

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The ITC experiments were performed using an iTC200 microcalorimeter (Malvern Panalytical, Malvern, UK). The antibodies were dialyzed separately in 50 mM HEPES‐NaOH (pH 7.5), 200 mM NaCl, 300 μM NaHCO3 at 4°C overnight. MtsA was concentrated using an ultrafiltration unit (Amicon‐Ultra‐0.5, 10K, Merck KGaA, Darmstadt, Germany) and diluted with 50 mM HEPES‐NaOH (pH 7.5), 200 mM NaCl, 300 μM NaHCO3, and the process was repeated three times. The cell was filled with 10 μM antibodies, and the syringe was filled with 100 μM MtsA. The first injection of 0.5 μL (omitted from the analysis) was followed by 19 injections of 2 μL with 120 s intervals at a constant temperature of 25°C. The titration syringe was continuously stirred at 750 rpm. The obtained data were fitted by nonlinear regression of the integrated data to a one‐site binding model using ORIGIN 7.0 software (MicroCal, Northampton, MA, USA).
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5

Purification and Characterization of EGFP Variants

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The EGFP-Y145L/S205V coding sequence in the pQE30 vector backbone (Qiagen, Hilden, Germany), with an N-terminal 6xHis tag, was expressed in the E. coli XL1 Blue strain (Invitrogen, Waltham, MA, USA) and purified using TALON metal affinity resin (Clontech, Kyoto, Japan) according to the manufacturer’s protocol. For the further fluorescence spectroscopy experiments, the spherical resin particles with the immobilized proteins were washed four times with phosphate-buffered saline (PBS pH 7.4, Gibco, Waltham, MA, USA) at a ratio of 1:20 v/v. The proteins were then solubilized using 100 mM imidazole, pH 8.0, and desalted by ultrafiltration with Amicon® Ultra-0.5 10 K (Merck, Tullagreen, Carrigtwohill Co Cork, Ireland) columns (the final buffer was PBS pH 7.4).
The time-resolved fluorescence spectroscopy of the purified protein was performed using a set of pH buffers in a pH range of 5.5–10.0. Each buffer contained 130 mM potassium gluconate, 20 mM sodium gluconate, 0.5 mM MgCl2, 0.2 mM EGTA and 30 mM Tris (pH 8.0–10.0), HEPES (pH 6.8–7.8) or MES (pH 5.5–6.6). Each specimen contained 5 μg/mL of EGFP-Y145L/S205V, dissolved in pH buffer at a ratio of 1:20 v/v.
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