Asymmetric PCR38 (link) was used to generate 83mer ssDNA substrates with heavy isotope labeled nucleotides. Each PCR reaction contained 1000 nM forward primer, 50 nM reverse primer, a 83mer synthetic template DNA (primer and template sequences are listed in Supplementary Table 1 ) and a dNTP-mixture in which one unlabeled dNTP was substituted with the respective heavy isotope-labeled dNTP (15N5-dGTP, 15N3-dCTP, 15N2-TTP, 13C10-dATP, or 15N5-dATP, Silantes). Amplification was performed in 50 cycles. Subsequently, excess primers and dNTPs were removed by Amicon Ultra-0.5 ml centrifugal filter units (Millipore) according to manufacturer’s instructions. The concentrated ssDNA was used for in vitro ADP-ribosylation and LC-MS/MS analysis as described below. Note, the 83mer ssDNA substrates contain a mixture of the respective heavy and natural nucleotide due to usage of unlabeled primer sequences.
Amicon ultra 0.5 ml centrifugal filter unit
Manufactured by Merck Group
Sourced in United States, Germany
The Amicon® Ultra-0.5 ml centrifugal filter unit is a laboratory device used for the concentration and purification of macromolecules such as proteins, peptides, and nucleic acids. It operates by filtering samples through a semi-permeable membrane during centrifugation, allowing the selective retention of the desired molecules while allowing smaller molecules to pass through.
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Lab products found in correlation
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Tecnai 10 transmission electron microscope, by Thermo Fisher Scientific (1 mentions)
Uranyl acetate, by Thermo Fisher Scientific (1 mentions)
Tla 100 fixed angle rotor, by Beckman Coulter (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Goat anti human igg peroxidase conjugate, by Merck Group (1 mentions)
0.2 μm syringe filter, by Merck Group (1 mentions)
Tgx stain free protein gel, by Bio-Rad (1 mentions)
Pierce rapid gold bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Human plasma, by Merck Group (1 mentions)
Ultracel 30 membrane, by Merck Group (1 mentions)
Genistein, by Merck Group (1 mentions)
Nitroblue tetrazolium, by Merck Group (1 mentions)
Melatonin, by Merck Group (1 mentions)
Aminoguanidine, by Merck Group (1 mentions)
2 4 dinitrophenylhydrazine, by Merck Group (1 mentions)
15 protocols using amicon ultra 0.5 ml centrifugal filter unit
1
Asymmetric PCR for Stable Isotope-Labeled DNA
2
Transmission Electron Microscopy of Extracellular Vesicles
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Transmission electron microscopy (TEM) was performed using EVs enriched from plasma samples. For EVs enriched using exoEasy, 20 μL of the elute was fixed with 5 μL of 4% formaldehyde solution. For EVs enriched using qEV, each 7-10 fraction pool was first concentrated to a final volume of 100-120 μl using 10kDa Amicon Ultra-0.5 mL Centrifugal Filter Units (Millipore, USA). After the concentration step, a 20 μL aliquot was fixed with 5 μL of 4% formaldehyde solution. Five μL EV aliquots from samples isolated by both methods were applied onto a Formvar/Carbon type B coated electron microscopy grid (Ted Pella Inc., USA) which was then washed with double distilled H2O, blotted dry with filter paper and stained with 2% uranyl acetate (Thermo Fisher Scientific, USA). The grid was again blotted dry and analyzed with a FEI Tecnai 10 transmission electron microscope (FEI, USA) at an accelerating voltage of 100 kV.
3
Milk Proteomic Analysis Preparation
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Milk sample preparation for proteomic analysis was carried out as described previously [17] . Briefly, milk was allowed to thaw at room temperature and centrifuged at 800 x g at 4°C for 10 min, the fat ring was removed, and skim milk was diluted 1:1 with lysis buffer, incubated at 95°C for 10 min and sonicated in a refrigerated water bath for 10 min, after which the suspension was centrifuged at 10.000 x g for 10 min at 4°C. Then, 7 µl of extract was subjected to filter-aided sample preparation (FASP) [18] (link). Protein samples were reduced, alkylated, and digested with trypsin on 3 kDa cut-off Amicon Ultra-0.5 mL centrifugal filter units (Millipore, Billerica, MA, USA). Peptide concentration was determined with a NanoDrop 2000 spectrophotometer (Thermo Scientific, San Jose, CA, USA).
4
Immunoprecipitation of TREM2 from BV2 Cells
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BV2 cells were grown to 80%-90% con uency in two dishes and treated with 20 µM S1P or not. After 2 h, the cells were washed thrice with PBS and collected with 400 µl homogenate buffer per dish (250 mM sucrose, 10 mM HEPES, 1 mM EDTA, 1 mM DTT, NaOH, pH to 7.4). The collected cells were frozen at -80 ℃ and underwent 5 freezing and thaw cycles to facilitate lysis. Then the buffer was homogenized further with bead mill. After centrifugation at 12000 rpm for 15 min at 4 ℃, the protein of cell lysates was acquired and incubated with 3 µl anti-TREM2 (abcam, ab125117) overnight at 4 ℃ on a rotating device, followed by adding 100 µl proteinA + G beads/ml lysate overnight to capture the conjugated polymersat 4 ℃ on a rotating device. Immunoprecipitates were collected by centrifugation at 8000 rpm for 2 min at 4 ℃ and washed thrice with 1 ml homogenate buffer, then resuspended in 50 mM NH 4 HCO 3 twice the volume of beads. After boiling, the supernatant was added 200 µl chromatographic grade ethaol, blended and centrifugated at 12000 rpm, 4 ℃ for 30 min to discard precipitation. The solution obtained was puri ed and concentrated with Amicon Ultra-0.5 ml Centrifugal Filter Units (Millipore), and detected by Analysis and Testing Center of Nanjing Medical University.
5
Labeling Tau Oligomers with Alexa Fluor 488
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Prepared recombinant TauO were provided by Dr. Rakez Kayed’s laboratory. They were produced and characterized following established and published protocols [33 , 34 (link)] and labeled (TauO488) as previously described [35 (link)–37 (link)]. Briefly, 1 mg of Alexa Fluor™ 488 NHS Ester Succinimidyl Ester (cat# A20000, Thermo Fisher Scientific) was dissolved in 0.1 M sodium bicarbonate to a final concentration 1 mg/ml, pH 8.3. The dye was then incubated with TauO in a 1:4 (w/w) ratio, rotating overnight at 4 °C on an orbital shaker. The following day, the solution was centrifuged (30 min, 15,000 × g) using 10-kDa Amicon Ultra‐0.5 ml Centrifugal Filter Units (cat# UFC501024; EMD Millipore, Burlington, MA, USA) to remove unbound dye. TauO were then washed with 1 × phosphate-buffered saline (PBS) until the flow-through solution was clear. The filter compartment was then flipped and centrifuged to collect the concentrate (2 min, 1000 × g). Oligomer concentrations were then quantified with the Pierce™ BCA Protein Assay Kit (cat# 23227, Thermo Fisher Scientific) and used for flow cytometry and immunofluorescence analyses.
6
Lipoprotein Fractions Purification by Ultracentrifugation
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Lipoprotein fractions were purified by isopycnic salt gradient ultracentrifugation (Figure 5 ) [3 (link),4 (link)]. Briefly, 0.9 mL of plasma sample, brought to d = 1.3 g/mL with solid NaBr (472.2 mg NaBr/mL plasma), were gently overlaid with 2.1 mL of a d = 1.006 g/mL solution (0.6% NaCl) (Figure 5 a), and centrifuged at 541,000× g for 3 h at 4 °C in a TL-100 series ultracentrifuge equipped with a TLA-100 fixed-angle rotor (Beckman Coulter, Indianapolis, IN, USA) (Figure 5 b). Afterwards, VLDL (d = 1.006–1.063 g/mL), LDL (d = 1.063–1.19 g/mL) and HDL (d = 1.19–1.21 g/mL) fractions were collected and further purified by a second centrifugation step, performed at 541,000× g for 2 h in saline solutions at density 1.006, 1.063, and 1.21 g/mL (Figure 5 c), respectively, followed by desalting and concentration using Amicon Ultra-0.5 mL centrifugal filter units (10 KDa MWCO, Merck-Millipore, Darmstadt, Germany). The degree of purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as previously described [3 (link)].
7
Quantifying CH25H in FLS cell lysates
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FLS lysates (20 µg of proteins) were obtained after 48 h and concentrated 3 times on Amicon® Ultra 0.5 mL centrifugal filter units (MWCO 3 kDa, Merck, Darmstadt, Germany) to exchange SDS with PBS. The concentration of CH25H in the lysates was determined by the human C25H/CH25H ELISA Kit (LS-F7759, LSBio, Seattle, WA, USA) as per the manufacturer’s protocol. The sensitivity was 49 pg/mL, and the intra-assay and interassay precision was below 10% and 12%, respectively.
8
SDS-PAGE and Immunoblotting of IgG
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SDS-PAGE was run using a 12% Mini-PROTEAN® TGX™ precast gel (Bio-Rad Laboratories, Inc., Hercules, CA, USA). For immunoblotting, a goat anti-human IgG peroxidase conjugate (Sigma-Aldrich, St. Louis, MO, USA) was used as a detection antibody at a 1:10,000 dilution. TMB Stabilized Substrate (Promega, Madison, WI, USA) was used for color development. Samples were concentrated using Amicon® Ultra 0.5 mL centrifugal filter units with a 10 kDa membrane (MilliporeSigma, Burlington, MA, USA). Prior to concentration, samples were filtered using a 0.2 μm syringe filter (MilliporeSigma, Burlington, MA, USA).
9
Preparation of Concentrated Conditioned Medium
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Passage 3 MEFs were seeded and maintained in 100 mm culture dish as above until confluent. Twenty-four hours before collection, culture medium was replaced with DMEM without phenol red and serum and was supplemented with 50 µg/mL ascorbic acid. Conditioned medium was collected after 24 h and concentrated 40-fold using Amicon Ultra-15 Centrifugal Filter Units (MilliporeSigma, UFC901024, Burlington, MA, USA). Buffer exchange with buffer containing 25 mM Tris-HCL pH8.0 and 25 mM NaCl was achieved using Amicon Ultra-0.5 mL centrifugal filter units (MilliporeSigma, UFC501008). Concentrated and buffer-exchanged medium were loaded into TGX stain-free protein gels (Bio-Rad, 17000927, Hercules, CA, USA) for blotting. The remaining attached cells were lysed with Pierce IP Lysis Buffer (ThermoFisher Scientific, 87788) containing complete EDTA-free Protease Inhibitor Cocktail (MilliporeSigma, 11873580001), and protein was quantified with Pierce Rapid Gold BCA Protein Assay kit (ThermoFisher Scientific, A53226).
10
Protein Binding Assay for CdG
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