Chondrocytes (5 × 104) were cultured in 12-well plates in 2 ml of DMEM containing 10% FCS for 2 days. Cells were rested in 1 ml of DMEM for 24 h, the medium was replaced with 1 ml of DMEM and used in the experiments. After incubation of various period of time, the medium was collected, concentrated 20-fold using spin filters (Microcon YM-30, Merck Millipore, Darmstadt, Germany) and 20 μl of 4× SDS-sampling buffer was added to 50 μl of each concentrated medium. The cells were lysed with 200 μl of 2× SDS-sampling buffer and 10 μl of samples were analyzed by SDS-PAGE under non-reducing conditions and Western blotting using anti-LRP1 α-chain, anti-LRP1 β-chain and anti-tubulin antibodies, respectively. Immune signals of LRP1 and tubulin were quantified using ImageJ, and the relative amounts of LRP1 α-chain in the medium and LRP1 α-and β-chains in the cell lysate were estimated within the linear range of measurements (Figure S1, A and B ) and normalized using tubulin and those in the standard cell lysates of chondrocytes as internal controls. An absolute number of LRP1 molecules released into medium were estimated in comparison of various concentrations of purified LRP1 within reasonable linear range.
Microcon ym 30
Manufactured by Merck Group
Sourced in United States, Germany
The Microcon YM-30 is a centrifugal ultrafiltration device designed for the concentration and desalting of macromolecular solutions. It utilizes a regenerated cellulose membrane with a molecular weight cut-off of 30,000 daltons to selectively retain molecules larger than the cut-off while allowing smaller molecules to pass through.
Automatically generated - may contain errors
Lab products found in correlation
Iodoacetamide iaa, by Merck Group (3 mentions)
Trypsin, by Promega (2 mentions)
Cy3mono nhs ester, by GE Healthcare (2 mentions)
Nanodrop nd ioo spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Amicon ultracel 30k filters, by Merck Group (2 mentions)
λ phosphatase, by New England Biolabs (1 mentions)
2 n morpholino ethanesulfonic acid mes, by Merck Group (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Dnase, by Merck Group (1 mentions)
Carbon coated copper grid, by Ted Pella (1 mentions)
Uranyl acetate, by Electron Microscopy Sciences (1 mentions)
Epigallocatechin gallate (egcg), by Merck Group (1 mentions)
1 2 dioleoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
Lb medium, by BD (1 mentions)
Thioflavin t tht, by Merck Group (1 mentions)
Sephacryl s 200, by Merck Group (1 mentions)
Isopropylthiogalactoside, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 5 3 carboxymethoxyphenyl 2 4 sulfophenyl 2h tetrazolium, by Merck Group (1 mentions)
Deae sephacel, by Merck Group (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
35 protocols using microcon ym 30
1
Quantification of LRP1 Release from Chondrocytes
2
Phosphatase Treatment of Immunoprecipitated Proteins
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Phosphatase treatments were performed on immunoprecipitated proteins by using λ phosphatase (New England Biolabs, Ipswich, MA) according to the manufacturer’s instructions. The control and phosphatase-treated samples were then concentrated using Microcon YM-30 centrifugal filter units (Merck-Millipore, France), analyzed by gel electrophoresis.
3
Quantification of LRP-1 Release from Chondrocytes
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Chondrocytes (5 × 104) were cultured in 12‐well plates in 2 ml of DMEM containing 10% FCS for 2 days. Cells were rested in 1 ml of DMEM for 24 hours, and the medium was replaced with 1 ml of DMEM and used in the experiments. After incubation for various periods of time, the medium was collected and concentrated 20‐fold using spin filters (Microcon YM‐30; Merck Millipore), and 20 µl of 4× SDS sampling buffer was added to 50 µl of each concentrated medium. The cells were lysed with 200 µl of 2× SDS sampling buffer, and 10 µl of samples were analyzed by SDS‐PAGE under nonreducing conditions and Western blotting using anti–LRP‐1 α‐chain, anti–LRP‐1 β‐chain, and antitubulin antibodies. Immune signals of LRP‐1 and tubulin were quantified using ImageJ software, and the relative amounts of LRP‐1 α‐chain in the medium and LRP‐1 α‐ and β‐chains in the cell lysate were estimated within the linear range of measurements (see Supplementary Figures 1A and B, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.40080/abstract ) and normalized using tubulin, and those in the standard cell lysates of chondrocytes were used as internal controls. An absolute number of LRP‐1 molecules released into medium was estimated by comparing various concentrations of purified LRP‐1 within a reasonable linear range.
4
Protein Purification and Characterization
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Carbon-coated copper grid was purchased from Ted Pella Inc (Redding, CA). DNase, DEAE-Sephacel, (−)-Epigallocatechin gallate (EGCG), isopropylthiogalactoside (IPTG), lysozyme, 2-(N-morpholino)ethanesulfonic acid (Mes), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeth-oxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), Sephacryl S-200, S-Sepharose, and thioflavin-T (Th-T) were purchased from Sigma-Aldrich (St. Louis, MO). 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) was obtained from Avanti Polar Lipids Inc (Alabaster, AL). LB medium was purchased from Becton Dickinson & Company (San Diego, CA), and uranyl acetate was obtained from Electron Microscopy Sciences (Hatfield, PA). Microcon YM30 was provided by Merck Millipore (Billerica, MA). Dimethyl cyclodextrin was supplied from the Microbial Carbohydrate Resource Bank at Konkuk University.
5
CSFV Recombinant Protein Analysis
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The supernatants (100 µL) were concentrated with a centrifugal filter (Microcon YM-30; Merck, Darmstadt, Germany) by centrifuge ×14,000 g at 25℃ for 30 minutes, then solubilized in 2× sample buffer and treated at100℃ for 5 minutes before use. The E2 CSFV recombinant protein (10 µL) was reduced with 2.5% β-mercaptoethanol. Proteins were either stained with Coomassie Brilliant Blue Stain (Thermo Scientific) or transferred to a polyvinylidene difluoride (PVDF) membrane (Invitrogen) for western blotting using Mini Trans-Blot cell system (BioRad, Hercules, CA, USA). The transferred PVDF membrane was blocked with 5% nonfat milk (Sigma-Aldrich) dissolved in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST). The membrane was incubated with mAb anti-CSFV antibody (The National Institute of Animal Health, Tsukuba, Japan) for 1 hour at 25℃, washed 3 times with PBST. Then, the membrane was incubated with POD-anti-mIgG (H+L) (GE Healthcare, Chalfont St. Giles, UK) for 1 hour at 25℃ and washed 3 times with PBST. Detection of immunoreactive bands was performed using Western Lightning Plus-ECL substrate (Thermo Scientific) as per the supplier's instructions.
6
Quantitative Proteome Profiling Using FASP
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Proteins were digested using the filter-aided sample preparation (FASP) method according to a previously described method with slight modifications (32 (link), 37 ). Briefly, the lysates (100 μg protein for each sample) were reduced with 10 mM DTT at 37 °C for 1 h and alkylated with 55 mM iodoacetamide (IAA, Sigma-Aldrich) at room temperature for 1 h in the dark. The alkylated lysates were transferred into the Microcon YM-30 centrifugal filter units (EMD Millipore Corporation), where the denaturing buffer was replaced by the 0.1 M triethylammonium bicarbonate (TEAB, Sigma-Aldrich, Saint Louis, MO), and then digested with sequencing grade trypsin (Promega) at 37 °C overnight. The resulting tryptic peptides were collected and labeled with 6-plex TMT reagents (Thermo Scientific) by incubating peptides with ethanol-dissolved TMT reagents for 2 h at room temperature in the dark. The labeling reaction was inactivated by the addition of 5% hydroxylamine, and the labeled samples were mixed together with equal ratios before being fractionated with reversed-phase (RP) high-performance liquid chromatography (HPLC).
7
Protein Denaturation and Digestion
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In total, 30 µg of protein extract was denaturated with 200 µL 8M urea on Microcon YM-30 (#MRCF0R030, Merck KGaA, Darmstadt, Germany) according to Wisniewski et al., in 2009 (FASP protocol), followed by reduction, alkylation and trypsin digestion as described in Frøyset et al. (2016) [29 (link)]
8
Protein Extraction and Digestion Protocol
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In the discovery cohort, the 8 samples of each group were mixed and aliquoted into 4 centrifuge tubes (see Fig. 1 ). The mixed samples were lysed in buffer containing 4% sodium dodecyl sulfate and 0.1 M Tris–HCl (pH 7.6). The protein concentration was determined by the BCA protein assay kit (Thermo Scientific, Rockford, IL). The proteins (100 μg) were incubated with 100 mM DTT at 37 ℃ for 1 h. After incubation, the lysates were transferred to a centrifugal filter (Microcon YM-30, EMD Millipore Corporation, Billerica, MA), and replaced with 200 μl UA (8 M urea, 100 mM Tris. Cl pH 8.5) twice. After the buffer was replaced, proteins within UA were alkylated with 55 mM iodoacetamide (IAA, Sigma–Aldrich, Saint Louis, MO) in a dark at room temperature for 15 min. The UA buffer was subsequently replaced with 0.1 M triethylammonium bicarbonate (TEAB, Sigma–Aldrich, Saint Louis, MO) and digested with trypsin (Promega, Madison, WI) (1:50 (w: w)) at 37 ℃ overnight. The desalting of resultant tryptic peptides was conducted by StageTips and stored at − 20 °C.
9
Proteomic Analysis of Decellularized Femoral Arteries
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Femoral arteries were harvested from New Zealand White rabbits. Three femoral arteries were decellularized using the ScCO 2 technique and three femoral arteries were decellularized using SDS-SDC detergents. Total protein was extracted from the decellularized graft. Protein samples were digested using the filter-aided sample preparation (FASP) method to generate tryptic peptides for LC-MS analysis. In brief, the samples were buffer exchanged with a molecular weight cut-off filter (Microcon YM-30; Merck Millipore Inc., Temecula, CA, USA), and the proteins were digested on the membrane by adding trypsin. All the digested peptides were collected and dried in a vacuum centrifuge tube. Prior to LC-MS analysis, all dried peptide mixtures were dissolved in 0.1% trifluoroacetic acid and desalted. LC-MS analysis was performed using a nanoACQUITY UPLC System (Waters Corporation, Milford, MA, USA) coupled with a high-resolution mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The mass spectrometer was operated in data-dependent mode with the following acquisition cycle: a full scan (m/z 350-1600) was recorded using an orbitrap analyzer at a resolution (R) of 240 000, and 10 peaks showing the maximum intensity and charge ≥2 were selected and fragmented using CID at a normalized collision energy of 35.
10
WSSV Capsid Protein Characterization
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Rod-shaped and oval-shaped WSSV capsids were suspended in SDS buffer [4% SDS and 100 mM tris (pH 7.5)], reduced with 10 mM dithiothreitol at 37°C for 1 hour, and transferred to the Microcon YM-30 (Millipore MRCF0R030, USA). The buffer was replaced with UA buffer [8 M urea and 100 mM tris (pH 8.5)], and proteins were alkylated with 55 mM iodoacetamide at room temperature for 15 min in the dark. The UA buffer was then replaced with 0.1 M triethylammonium bicarbonate solution (pH 8.5). Proteins were digested with sequencing grade trypsin at 37°C overnight, and the elution was collected by ultrafiltration with the cutoff at 5000 Da. Peptides were desalted by strong cation exchange StageTips and concentrated by a SpeedVac vacuum concentrator (Thermo Fisher Scientific, USA).
Peptides were analyzed on a TripleTOF 5600 mass spectrometer (AB Sciex, USA) coupled with an Eksigent NanoLC-1D platform assembled in information-dependent mode. Peptides were identified from MS/MS spectra using ProteinPilot version 4.2 (Applied Biosystems, USA) by searching against the protein sequences of WSSV. The fixed modification was carbamidomethylation of cysteine residues. Trypsin was specified as the proteolytic enzyme with two missed cleavages. Mass tolerance was set to 0.05 Da, and the maximum false discovery rate for proteins and peptides was 1%.
Peptides were analyzed on a TripleTOF 5600 mass spectrometer (AB Sciex, USA) coupled with an Eksigent NanoLC-1D platform assembled in information-dependent mode. Peptides were identified from MS/MS spectra using ProteinPilot version 4.2 (Applied Biosystems, USA) by searching against the protein sequences of WSSV. The fixed modification was carbamidomethylation of cysteine residues. Trypsin was specified as the proteolytic enzyme with two missed cleavages. Mass tolerance was set to 0.05 Da, and the maximum false discovery rate for proteins and peptides was 1%.
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