Native MS samples were prepared by exchanging the buffer in which purified VLPs were obtained for aqueous ammonium acetate using multiple concentration and dilution steps with a centrifuge filter (Millipore, Billerica, MA). For the experiments shown in Fig. 1 , samples were prepared in 100 mM ammonium acetate (pH 7.4) and kept on ice. For control experiments under HDX conditions, samples were heated at 60°C in water and spectra were acquired at multiple time points. Before injection into the mass spectrometer, low amounts (<25 mM) of triethylammonium acetate were added. MS experiments were performed on a modified QToF II (MS Vision, Waters, Elstree, United Kingdom) (40 (link)), operating at 10 mbar source pressure, 1300–1500 V capillary voltage, 100 V cone voltage, and 300 V collision energy with 2 × 10−2 mbar pressure in the collision cell using xenon as the collision gas (41 (link)). Tandem MS experiments were acquired by full envelope selection and fragmentation using a 400 V potential over the collision cell. Aliquots of the samples (at ∼2 μM monomer concentration) were introduced in the mass spectrometer through nano-electrospray ionization using gold-coated boro-silicate capillaries produced in house. All data were analyzed using Masslynx 4.1 software (Waters).
Centrifuge filter
Manufactured by Merck Group
Sourced in United States
A centrifuge filter is a laboratory equipment used to separate solid particles or cells from a liquid solution through the application of centrifugal force. It functions by spinning the sample at high speeds, causing the denser components to settle at the bottom of the container, allowing the liquid to be filtered or decanted.
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Lab products found in correlation
Masslynx 4, by Waters Corporation (1 mentions)
Transit x2 dynamic delivery system, by Mirus Bio (1 mentions)
Quant it protein assay kit, by Thermo Fisher Scientific (1 mentions)
Tissuelyser lt, by Qiagen (1 mentions)
Fibronectin, by Abcam (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
4t1 cell, by American Type Culture Collection (1 mentions)
0.45 μm filter, by Merck Group (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
3xflag peptide, by Merck Group (1 mentions)
Bn page, by Thermo Fisher Scientific (1 mentions)
Anti flag affinity gel, by Selleck Chemicals (1 mentions)
Skbr3, by American Type Culture Collection (1 mentions)
Hexane, by Sinopharm (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Sw620, by American Type Culture Collection (1 mentions)
Ethanol, by Sinopharm (1 mentions)
N 3 dimethylaminopropyl n ethylcarbodiimide hydrochloride, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Sinopharm (1 mentions)
11 protocols using centrifuge filter
1
Native Mass Spectrometry Analysis of Virus-Like Particles
2
Enzymatic Conversion of 2-OPP to 2-HPP
3
Surface Modification of Single-Walled Carbon Nanotubes
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For the surface modification of SCNTs, detailed methods are again provided in our prior publication [13 (link)]. Briefly, a short chain PEG linker (NH2-PEG-COOH, MW, 2 K, Sigma-Aldrich, St. Louis, MO, USA) was selected for conjugation through the EDC/Sulfo-NHS method. The EDC/NHS–activated SCNT was conjugated with a PEG linker. The resultant PEG-SCNT was again separated and purified using a centrifuge filter (molecular weight cutoff of 10 KDa, Millipore, Burlington, VT, USA) three times to remove excess PEG. PEG-SCNT products were dried under vacuum for analysis and conjugation with V1V2 proteins via an EDC/NHS reaction. Thermal Gravimetric Analysis (TGA) curves of PEG-SCNT and SCNT alone were used to confirm and quantify the coating of functionalities (linkers) on the SCNT surfaces. The size of CNT samples was measured using DLS and SEM to ensure the obtention of proper dimensional SCNTs.
4
Multicolor Dye Preparation for Calcium Imaging
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A glass pipette (1.2 mm OD with filament, World Precision Instruments) was pulled to a tip diameter of 4 ~ 5 μm by a pipette puller (P-97, Sutter Instruments). Oregon Green 488 BAPTA-1 AM (OGB-1 AM) was dissolved in DMSO with 20% pluronic acid. Sulphorhodamine (SR-101) was added to distinguish astrocytes from neurons40 (link). SR-101 was useful for visualization of the dye ejection as well. This solution was then diluted in a pipette solution containing (in mM) 150 NaCl, 2.5 KCl and 10.0 HEPES, with its pH adjusted to 7.4 with KOH. The final concentrations were 0.8 mM for OGB-1 AM and 0.05 mM for SR-101. All chemicals were obtained from Molecular Probes or Sigma.
The final dye solution was sonicated for 10 minutes and filtered with a centrifuge filter (0.45 μm pore size, Millipore).
5
Evaluation of IDUA Gene Delivery Plasmids
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H2.35 (a mouse liver cell line) and Huh7 (a human hepatoma cell line) (ATCC, Manassas, VA) cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and cultured at 37°C with 5% CO2. The pAAV.TBG.hIDUA, pAAV.TBG.hIDUA-MTfp, pAAV.TBG.MTfp-hIDUA, and pAAV.TBG.EGFP plasmids were transfected into H2.35 or Huh7 cells using the TransIT-X2 Dynamic Delivery System (Mirus, Madison, WI) according to the manufacturer’s recommendations. 48 h after transfection, cell lysates and culture supernatants were harvested for an IDUA enzyme activity assay and western blot analysis, and culture supernatants were concentrated using a centrifuge filter (Millipore, Temecula, CA) prior to western blot analysis.
6
Excretory-Secretory Products from Tapeworms
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In order to obtain excretory-secretory products (ESP), the tapeworm larvae were removed from the peritoneal cavity of a female ICR mouse 3 months after infection, washed 5 times in sterile PBS, and placed in 75 cm2 culture flasks (Eppendorf) with DMEM (Dulbecco’s Modified Eagle’s Medium – high glucose, Sigma-Aldrich) supplemented with 100 U/ml of penicillin-streptomycin. The number of larvae per flask was roughly 300 cysticerci for T. crassiceps and 2,000 tetrathyridia for M. corti. The tapeworm larvae were then cultured at 37°C and 5% CO2. Every two days, the medium was aspirated, filtered through a 0.22 μm syringe-mounted filter (Sigma-Aldrich), frozen at -80°C and replaced with a new dose for a total of 14 days, with the medium collected after the first two days being always discarded. The collected medium was then concentrated using a centrifuge filter (Merck-Millipore). For in vitro assays with B16F10 cells, the ESPs were separated into fractions according to protein size (<3, 3-10, 10-30, >30 kDa) using centrifuge filters (Merck-Millipore). The concentration of total proteins was determined using a Quant-iT™ Protein Assay Kit (Invitrogen).
7
Plasma Amino Acid Profiling
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To remove the protein components in the collected plasma samples, equal amounts of 3% sulfosalicylic acid were mixed with each sample and the supernatant was collected after centrifugation. The sample pH was adjusted to pH 2-3 and then filtered using a centrifuge filter (Merck). The whole process was operated on ice. The amino acid composition was measured using JLC-500/V2 Automatic Amino Acid Analyzer (JEOL).
8
Quantifying Fibronectin Levels in Frozen Skin Samples
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Frozen skin samples (4 × 4 mm) were homogenized using a TissueLyser LT (Qiagen, Germantown, MD, USA) in ice-cold 7M Urea buffer containing protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO, USA). Samples were homogenized, and sample buffer was exchanged to PBS using centrifuge filters (10 kDa, Millipore, USA). Total protein content was measured by Bradford Protein Assay, and samples were normalized to total protein for Western blots experiments. Samples were run on 10% polyacrylamide gels, transferred to nitrocellulose membranes, and probed for fibronectin (1:1000 dilution; Abcam, Cambridge, MA, USA) as previously described (Hendel & Granville, 2013 (link)). GAPDH was probed as a loading control. Antibody detection was achieved using the Odyssey Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE, USA), and densitometry was performed with image j software.
9
Characterization of 4T1 and EO771 Cell Lines
10
VCP Purification and Characterization
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The pCMV-VCP plasmids were transfected to H1299 cells using PEI and cells were collected after 24-48 hours transient overexpression. U2OS cells stably expressing Flag- or mCheery-VCP were directly cultured and harvested for affinity or gel filtration purification. Cells were lysed with buffer A (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% Triton X-100, and 10% glycerol), supplemented with Complete protease inhibitor cocktail tablet (Roche Applied Science). Anti-Flag Affinity Gel (Bimake) was used for affinity purification. After being extensively washed, Flag-VCP was eluted with buffer B (50 mM Tris-HCl pH7.4, 150 mM NaCl,100 μg/ml 3x FLAG peptide (Sigma Aldrich)). The dodecamer species were enriched by collecting earlier elution fractions in gel filtration as shown in Figure S1 E. Samples were concentrated and buffer-exchanged to buffer C (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM tris (2-carboxyethyl) phosphine (TCEP)) or buffer D (20 mM Hepes pH7.4, 150 mM NaCl and 1 mM TCEP) using centrifuge filters (Millipore) for SDS-PAGE, blue native PAGE (BN-PAGE, Thermo Fisher) and cryo-EM analyses.
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