To express plasmids in neurons, constructs were cloned into lentiviral backbones and co‐transfected with pMD2.G (Addgene, #12259) lentiviral envelope and with pCMV∆R8.2 (Addgene, #12263) Lentiviral packaging into HEK cells. One day after transfection, medium was changed for X10 Opti‐MEM(R) (Gibco) supplemented with 100× Pen/Strep (Gibco). Forty hours after transfection, conditioned medium was harvested and spun down at 1,000 g to remove cell debris. Supernatant was transferred to Amicon spin filters (Millipore, 100 kDa cutoff) and spun down at 4,000 g for 30 min. Centrifugation was repeated until all supernatant passed the filter. Concentrated virus was diluted in PBS and filtered with a 0.2 μm filter (VWR). Virus was stored at −80°C until use.
Amicon spin filter
Manufactured by Merck Group
Sourced in Germany, United States
Amicon spin filters are laboratory centrifugation devices used for the separation, concentration, and purification of macromolecules, such as proteins, nucleic acids, and other biomolecules, from complex mixtures. They employ a membrane-based filtration system to selectively retain target analytes while allowing smaller molecules and solvents to pass through.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Pcmvr8, by Addgene (1 mentions)
0.2 μm filter, by Avantor (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Micro bca assay kit, by Thermo Fisher Scientific (1 mentions)
High performance liquid chromatography grade solvents, by Thermo Fisher Scientific (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
Ms grade trypsin lys c, by Promega (1 mentions)
Protein g sepharose, by Abcam (1 mentions)
Peakcal device, by Malvern Panalytical (1 mentions)
Microcal evaluation software, by Malvern Panalytical (1 mentions)
Trifluoromethanesulfonic acid, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Ethanolamine, by Merck Group (1 mentions)
Diethyl ether, by Merck Group (1 mentions)
Superdex 200 pg column, by GE Healthcare (1 mentions)
Polycarbonate transwell inserts, by Corning (1 mentions)
21 protocols using amicon spin filter
1
Efficient Lentiviral Transduction in Neurons
2
Purification and Characterization of NHE1 Protein
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NHE1680-815 was produced using M9 medium as previously described [29 (link),30 (link)]. NHE1680-815 was exchanged into HEPES buffer as above using Amicon spin filters (Millipore, Darmstadt, Germany). NMR spectra were recorded at 10 °C.
3
Proteomics Analysis of Extracellular Vesicle Proteins
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Ultrapure-grade 2,2,2-trifluoroethanol, trifluoroacetic acid, iodoacetamide and dithiotreitol (DTT) were from Sigma-Aldrich. High-performance liquid chromatography-grade solvents (methanol, acetonitrile and water) and formic acid were from Fisher Scientific. MS-grade trypsin/Lys-C was from Promega (Madison, WI). Amicon spin filters, 0.5 ml, 3 kDa molecular weight cut-off (MWCO), were from Millipore. Solid-phase extraction C18 tips were from Agilent. A measure of 4 ml of EPS urine was concentrated to ∼500 μl using a spin filter with a molecular weight cutoff of 3 kDa, and proteins were precipitated overnight by the addition of ice-cold 100% methanol. Protein pellets were washed twice with 100% methanol and air-dried. Protein resolubilization was performed by the addition of 50% 2,2,2-trifluoroethanol at 60 °C for 2 h. Following reduction with DTT and alkylation with iodoacetamide, proteins were digested overnight at 37 °C using 2 μg trypsin/Lys-C. The reaction was quenched by the addition of trifluoroacetic acid. Desalting was performed by solid-phase extraction using C18 tips. Solvents were removed by vacuum centrifugation, and peptides were resolubilized in 5% acetonitrile and 0.1% formic acid. Peptide concentrations were determined by the micro-BCA assay kit (Thermo Fisher Scientific).
4
Purification of Conjugation Reactions
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Crude conjugation reactions were purified by SEC using a BioLogic FPLC (Bio-Rad, Hercules, CA, USA) and a SEC650 10 × 300 gel filtration column (Bio-Rad) equilibrated in PBS with a flow rate of 1.8 mL/min. Absorbance at 230, 260 and 280 nm was measured and 0.5 mL fractions were collected across all significant peaks. The highest purity fractions were pooled and concentrated using Amicon spin filters, 30 kDa MWCO (Millipore, Burlington, MA, USA) and quantified by BCA protein assay (Thermo Fisher Scientific).
5
Purification of NMO-IgG from Plasmapheresis
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AQP4 antibody (NMO-IgG) was among the total IgG from plasmapheresate donated by aquaporin-4 antibody-seropositive donors undergoing treatment for a relapse; fluids were stored at −80°C until use. Plasmapheresate was diluted 3-fold in PBS, clarified at 10,000 ×g for 15 minutes at 4°C, and filtered with 70 μm strainer (Millipore) to remove particulate materials. Clarified solution was loaded on to 8 ml columns of Protein G-Sepharose (Biovision Inc., cat. no. 6511-25) equilibrated with phosphate buffered saline (PBS). Columns were then washed with 10 column volumes (CV) of PBS. Purified IgG was eluted with 0.1 M glycine-HCl, pH 2.6 into 1.5 ml tubes containing 90 μl of neutralization solution (1 M Tris-HCl pH 9.0). Fractions with 280 nm absorbance greater than 5-fold over background were pooled and buffer-exchanged to PBS using Amicon spin filters (50 KDa MWCO, 15 ml; Millipore). The final volume was adjusted to 7–10 ml of PBS and sterile filtered. The final yield typically was 140–160 mg of IgG from 50 ml of plasmapheresate.
6
Antibody-oligonucleotide Conjugation Methods
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Ab-oligo conjugates were generated using both non-specific and site-specific conjugation methods. Except where noted, conjugates were purified using 0.5 ml 100 kDa Amicon spin filters (MilliporeSigma Burlington, MA). DBCO reagents were diluted from dimethyl sulfoxide (DMSO) stocks to maintain < 1% DMSO in the presence of antibody to minimize degradation. Conjugates were resuspended in 1X PBS, pH 7.4 and absorbance was measured by spectroscopy to estimate the concentrations of oligo and antibody.
7
Secretome Profiling of ER Stress
8
Isothermal Titration Calorimetry of Protein Complexes
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To analyze the complex formation in vitro, isothermal titration experiments were performed using a Malvern PEAKCal device. All samples were prepared by dialysis overnight against the standard measuring buffer and concentrated with Amicon spinfilters (Merck Millipore, Burlington, MA, USA) to adjust concentrations. The standard assay settings were 750 rpm for stirring, with 5 µW used as the reference power; after 60 s of initial delay, the injection process started with 0.4 µL and an injection duration of 0.8 s. The first injection was followed by 18 injections with 2 µL and a duration of 4 s with 150 s of spacing. For all titrations, 300 µL of 4 µmol/L SubA was titrated with 20 µL of 320 µmol/L SubB solution in 19 steps at 25 °C. Evaluation of the titrations was performed with the MicroCal evaluation software from Malvern (Worcestershire, UK).
9
Antibody Purification and Characterization
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In this study we have
used a humanized IgG4 antibody against trinitrophenyl (TNP). The antibody
was manufactured by Novo Nordisk A/S and purified using Protein A
chromatography, and subsequently concentrated and buffer exchanged
into a 10 mM Histidine, 10 mM NaCl, pH 6.5 buffer at a concentration
of 100 mg/mL. From this stock solution, samples were prepared by concentrating
and buffer exchanging into either 20 mM Histidine, 10 mM NaCl, pH
6.5 or 20 mM Histidine, 50 mM NaCl, pH 6.5 buffers using Amicon spin
filters with a molecular weight cutoff at 100kD (Merck, Germany).
The two different solvents thus have a total ionic strength of either
17 mM or 57 mM, respectively. Samples with decreasing concentration
were then prepared from the concentrated sample by dilution, determining
the antibody concentration using UV/vis absorption at 280 nm and a
molecular extinction coefficient derived from the amino acid composition
of 223400 M–1 cm–1 (E0.1%,1 cm280 nm = 1.489 g–1 L cm–1).
used a humanized IgG4 antibody against trinitrophenyl (TNP). The antibody
was manufactured by Novo Nordisk A/S and purified using Protein A
chromatography, and subsequently concentrated and buffer exchanged
into a 10 mM Histidine, 10 mM NaCl, pH 6.5 buffer at a concentration
of 100 mg/mL. From this stock solution, samples were prepared by concentrating
and buffer exchanging into either 20 mM Histidine, 10 mM NaCl, pH
6.5 or 20 mM Histidine, 50 mM NaCl, pH 6.5 buffers using Amicon spin
filters with a molecular weight cutoff at 100kD (Merck, Germany).
The two different solvents thus have a total ionic strength of either
17 mM or 57 mM, respectively. Samples with decreasing concentration
were then prepared from the concentrated sample by dilution, determining
the antibody concentration using UV/vis absorption at 280 nm and a
molecular extinction coefficient derived from the amino acid composition
of 223400 M–1 cm–1 (E0.1%,1 cm280 nm = 1.489 g–1 L cm–1).
10
Azido-PEG Conjugation and Fluorescent Labeling
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Solvents and reagents were purchased from commercial suppliers and used without further purification unless otherwise mentioned. Trifluoromethanesulfonic acid, benzene (dry), methylene chloride (dry), diethyl ether, dimethyl sulfoxide (DMSO), acetonitrile (ACN, HPLC grade), ethanol amine, 3,6-dioxa-1,8-diaminooctane, DBCO-PEG12-maleimide and phosphate-buffered saline (PBS) tablets were purchased from Sigma-Aldrich. Azido poly (ethylene glycol) methyl ether (azide-PEG113-OH) was purchased from Rapp Polymere, 5(6)-tetramethyl rhodamine (TMR) cadaverine was purchased from Biotium, and the peptide sequence was obtained from GenScript. 1-(4-(aminomethyl)-benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (IMDQ) was synthesized as reported here [59 (link),60 (link),61 (link)] and 5-methyl-5-pentafluorophenyloxycarbonyl-1,3-dioxan-2-one (MTC-PFP) was synthesized as reported here [48 (link)]. Millipore water was prepared using a MILLI-Q R Reference A+ system at a resistivity of 18.2 MΩ·cm and total organic carbon of <5 ppm. Dialysis was performed using Spectra/Por® 7 dialysis membranes with a molecular weight cutoff of 1000 g mol−1, and Amicon spin filters were used with a molecular weight cut-off (MWCO) of 10,000 g mol−1 obtained from Merck Millipore.
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