All procedures for sample preparation were performed at room temperature. CM (9 ml per sample) was concentrated with 3 kDa MWCO Amicon Ultra Centrifugal filter devices (Merck Millipore) to a final volume of 30 μl. Protease inhibitors were added, and protein concentration was determined with the Bradford Assay. The concentrated samples were processed with the filter-aided sample preparation (FASP) method as described previously [30 (link)], with minor modifications [31 (link)]. Briefly, 200 μg of each sample was mixed with lysis buffer (0.1 M Tris–HCl pH 7.6 supplemented with 4% SDS and 0.1 M DTE) and buffer exchange was performed in Amicon Ultra Centrifugal filter devices (0.5 ml, 30 kDa MWCO; Merck Millipore) at 14,000×g for 15 min. Each sample was then diluted with urea buffer (8 M urea in 0.1 M Tris–HCl pH 8.5) and centrifuged. The concentrate was diluted again with urea buffer and centrifugation was repeated. Alkylation of proteins was performed with 0.05 M iodoacetamide in urea buffer for 20 min in the dark followed by centrifugation at 14,000×g for 10 min. Additional series of washes were conducted with urea buffer (2 times) and 50 mM NH4HCO3 pH 8.5 (2 times). Tryptic digestion was performed overnight in the dark, with a trypsin-to-protein ratio of 1:100. Peptides were eluted by centrifugation at 14,000×g for 10 min, lyophilized and stored at –80 °C until further use.
Amicon ultra centrifugal filter device
Manufactured by Merck Group
Sourced in United States, Germany, Ireland
Amicon Ultra centrifugal filter devices are high-performance membrane-based filtration units designed for the rapid concentration and purification of biological samples. These devices utilize a centrifugal force to efficiently separate molecules based on their size, allowing for the concentration and recovery of target analytes from complex mixtures.
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Lab products found in correlation
Ni nta column, by Qiagen (5 mentions)
Captureselect c tagxl affinity matrix prepacked column, by Thermo Fisher Scientific (3 mentions)
Elutrap electroelution system, by Cytiva (3 mentions)
Histrap high performance 1 ml columns, by GE Healthcare (3 mentions)
Hitrap heparin columns, by Merck Group (2 mentions)
Pet32a expression vector, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Histrap hp column, by GE Healthcare (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Protein g sepharose, by GE Healthcare (2 mentions)
Superfect transfection reagent, by Qiagen (2 mentions)
Sequagel, by National Diagnostics (2 mentions)
Taqman pcr amplification, by Thermo Fisher Scientific (2 mentions)
Benzonase, by Merck Group (2 mentions)
Endosafe pts 2005, by Charles River Laboratories (1 mentions)
Rotavapor r 215, by Büchi (1 mentions)
Slide a lyzer dialysis cassette, by Thermo Fisher Scientific (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
95 protocols using amicon ultra centrifugal filter device
1
Filter-Aided Sample Preparation for Proteomic Analysis
2
Production and Titration of AAV Vectors
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AAV vectors were produced as described previously26 (link)72 (link). Briefly, AAV virions were produced that contained a 1:1 ratio type 1 and type 2 capsid proteins by transfecting human embryonic kidney (HEK) 293 cells with the AAV backbone plasmids pAM-FLEX-TeLC or pAM-FLEX-GFP26 (link) and AAV1 (pH21), AAV2 (pRV1) helper plasmids along with the adenovirus helper plasmid pFdelta6 using the calcium phosphate method72 (link). 48 hours post transfection the cells were harvested and AAVs purified using 1 ml HiTrap heparin columns (Sigma) and concentrated using Amicon Ultra centrifugal filter devices (Millipore). Infectious AAV particles (viral titre) were calculated by serially infecting HEK293 cells stably expressing Cre-recombinase26 (link) and counting GFP-positive cells.
3
IgG Purification via Protein G Chromatography
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IgGs were purified from 1 to 2 mL of plasma samples by gravity-flow protein G affinity chromatography (GE Healthcare). The elution fraction was then desalted and concentrated via multiple round centrifugations with Amicon Ultra centrifugal filter devices (Millipore) in normal saline solution. IgG concentration was measured with BN ProSpec analyzer (Siemens).
4
PLGA and PCL Nanoparticle Synthesis
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PLGA and PCL nps were prepared by an oil-in-water (o/w) emulsion solvent evaporation method as described previously [34 (link)]. Briefly, 400 μl of mixture of PLGA or PCL (4 mg) and PEGPE (8 mg) in chloroform was added dropwise into 4 ml water. Then the solution was sonicated at 10 W (Branson Sonifier 450 microtip probe ultrasonicator, Danbury, CT, USA) for four 25s bursts interspersed with cooling on an ice bath for 60s. The chloroform was separated from the emulsified solution by using a rotary evaporator (Rotavapor R-215, Büchi, Postfach, Switzerland). Nanoparticles were washed three times by centrifugation (18000 g, 5 mins) using Amicon ultra centrifugal filter devices (Millipore, Billerica, MA, USA) to remove free PEGPE. Endotoxin level in PLGA and PCL nps were analyzed using LAL-based assay Endosafe PTS-2005 (Charles River Laboratories, Wilmington, MA) and were found to be < 2.5 EU mL-1.
5
Affinity Purification and Interaction Studies
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GST-fused and MBP-fused proteins were purified from E. Coli by affinity chromatography with Glutathione Sepharose 4B and amylose resin, respectively, as described in the instruction manuals (GE Healthcare and New England Biolabs, respectively). Purified GST-fused and MBP-fused proteins were dialyzed using Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific) and were concentrated by Amicon Ultra Centrifugal filter devices (Millipore, Billerica, MA) and were separated by electrophoresis on a 10% SDS-PAGE and visualized by Coomassie Brilliant Blue (CBB) staining (Simply Blue SafeStain, Thermo Fisher Scientific).
For direct interaction studies, MBP-fused proteins were incubated with amylose resin at 4°C for 2 hours and washed with PBS. Purified GST-fused proteins were added to amylose-resin-bound MBP-fused proteins, and the mixture was incubated overnight at 4°C. After washing, the bound proteins were eluted with NuPAGE LDS sample buffer at 99°C for 10 minutes.
Twenty five microgram of purified MBP-LZTFL1 fusion protein was cleaved in 20 mM Tris-HCl, 100 mM NaCl, 2 mM CaCl2 (pH 8.0) containing 1 μg of Factor Xa (New England Biolabs) for 6 hours at room temperature and used for direct interaction studies with purified TfR1-Myc-Flag protein (TP326147, OriGene) conjugated anti-TfR1 antibody beads or GST-fused β1subunit of AP-1 conjugated Glutathione Sepharose 4B.
For direct interaction studies, MBP-fused proteins were incubated with amylose resin at 4°C for 2 hours and washed with PBS. Purified GST-fused proteins were added to amylose-resin-bound MBP-fused proteins, and the mixture was incubated overnight at 4°C. After washing, the bound proteins were eluted with NuPAGE LDS sample buffer at 99°C for 10 minutes.
Twenty five microgram of purified MBP-LZTFL1 fusion protein was cleaved in 20 mM Tris-HCl, 100 mM NaCl, 2 mM CaCl2 (pH 8.0) containing 1 μg of Factor Xa (New England Biolabs) for 6 hours at room temperature and used for direct interaction studies with purified TfR1-Myc-Flag protein (TP326147, OriGene) conjugated anti-TfR1 antibody beads or GST-fused β1subunit of AP-1 conjugated Glutathione Sepharose 4B.
6
Recombinant Mouse LCN2 Production
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The coding sequence for mouse LCN2 cDNA was amplified from brain cDNA by PCR using Ex Taq reagents. The primer sequences used to amplify mouse Lcn2 were 5′-AAACCATGGCCCTGAGTGTCAT-3′ and 5′-GCCTGAACCATTGGGTCTCTGC-3′. The PCR products were subcloned into the pCR-4 vector according to the manufacturer’s protocol and sequenced. The mouse LCN2 cDNA was then ligated into the pcDNA3.1 expression vector. Human embryonic kidney (HEK293) cells were cultured in a six-well dish with DMEM containing 10 % heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5 % CO2. Plasmid DNA was transfected into HEK293 cells with the FuGENE HD transfection reagent (Promega, Madison, WI) following the manufacturer’s protocol. The culture media were exchanged to serum-free media 24 h after transfection and was collected 48 h after transfection. Five hundred microliters of culture medium were further centrifuged at 14,000 × g for 10 min at 4 °C for concentration and was replaced with phosphate-buffered saline (PBS) using Amicon Ultra Centrifugal Filter Devices (10 K molecular weight cutoff, Millipore, Billerica, MA, USA). The buffer exchange was repeated three times. The concentrated media, the final volume of which was approximately 60 μL, were prepared for immunoblot and glycosylation analyses.
7
Purifying Ca2+-free EhCaBPs for ITC
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Five different EhCaBPs (EhCaBP1, 3, 5, 6, and 7) were overexpressed and purified as described earlier [36] , [37] (link). In order to obtain accurate measurements of Ca2+-binding energetics, it was essential to have the protein in its apo-form with no contamination of Ca2+ in the buffers. Hence, all of the buffers used for isothermal titration calorimetry (ITC) were decalcified using Chelex 100 resin (Bio-Rad). Decalcified ITC buffer (100 mMNaCl and 50 mM Tris-Cl, pH 7.0) was prepared by treatment with Chelex 100 resin (Bio-Rad). Each protein solution was treated with 5 mM EGTA and 2 mM EDTA to remove Ca2+ and Mg2+. The EDTA/EGTA bound to metal ions were removed from protein solution using Amicon ultra centrifugal filter devices (Millipore), through extensive buffer exchange (decalcified). Before the ITC experiment, the sample cell and injection syringe of the ITC machine (Microcal Inc.) were extensively cleaned using the decalcified buffer.
8
Knockdown of CALR in AcPP cells
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Three short-hairpin RNAs (shRNA) targeting CALR (shCALR1: GCGGCCTGATAATACCTAT, shCALR2: GCTGGATCGAATCCAAACA, and shCALR3: GCTGGATCGAATCCAAACA) were synthesized by GenePharma Biotechnology (Shanghai, China). The 293 T-cells were transiently transfected with the recombinant lentiviral shRNA constructs or empty carrier (pLVTHM as negative control) along with the pCMV∆8.9 and pMD2G plasmids in a ratio of 2:2:1 using Lipofectamine 3000 (Invitrogen, United States) according to the manufacturer’s protocol. Virus-containing supernatants were collected at 24 and 48 h after transfection, pooled together, and then concentrated by centrifugation (5,000 g, 40 min) using the Amicon ultra centrifugal filter devices (Millipore Corporation, United States). Then, the AcPP cells were infected with the lentiviruses carrying shCALR1, shCALR2, shCALR3, or the negative control in the presence of 5 μg/ml polybrene (Sigma-Aldrich). The GFP expressing cells were sorted by flow cytometry (BD FACSAria, United States) according to the manufacturer’s manual. The efficiency of CALR silencing by these shRNAs was determined using Western blotting and qRT-PCR assays.
9
Comprehensive Western Blot Protocol
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Western blotting was performed as described previously (34 (link)–36 (link)). Cytoplasmic and nucleic extracts of cells or liver tissues were separated using the protein extraction kit (Beyotime, Shanghai, China) following the manufacturer’s protocols. Intracellular proteins in cells or liver tissues were extracted using cold lysis buffer (25 mM Tris-HCl, PH 7.6, 150 mM NaCl, 1% NP-40, 0.1% SDS) plus protease inhibitor cocktail and phenylmethanesulfonyl fluoride (PMSF) on ice. The supernatant proteins were concentrated from the cultured media using Amicon Ultra Centrifugal Filter Devices (Millipore, USA) according to the manufacturer’s instructions. The collected protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore). Membranes for supernatant proteins were stained with Ponceau S (Sigma-Aldrich) as the loading control. Subsequently, membranes were blocked for 1 h with nonfat dry milk solution (5% in TBS) containing 0.1% Tween-20. The blots were probed subsequently with the indicated primary antibodies and the corresponding secondary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence (Yeasen, Shanghai, China). The antibodies used in this study were listed in Supplementary Table 2
10
Purification of SARS-CoV-2 Spike Protein Variants
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The recombinant proteins named rS1WT (Wuhan) and rS1 B.1.351 were purified using a CaptureSelectTM C-tagXL Affinity Matrix prepacked column (ThermoFisher) and followed the manufacturer’s guidelines [80 (link)]. Briefly, the C-tagXL column was conditioned with 10 column volumes (CV) of equilibrate/wash buffer (20 mM Tris, pH 7.4) before sample application. Supernatant was adjusted to 20 mM Tris with 200 mM Tris (pH 7.4) before being loaded onto a 5-mL prepacked column per the manufacturer’s instructions at 5 ml/min rate. The column was then washed by alternating with 10 CV of equilibrate/wash buffer, 10 CV of strong wash buffer (20 mM Tris, 1 M NaCl, 0.05% Tween-20, pH 7.4), and 5 CV of equilibrate/wash buffer. The recombinant proteins were eluted from the column by using elution buffer (20 mM Tris, 2 M MgCl2, pH 7.4). The eluted solution was concentrated and desalted with preservative buffer (PBS) in an Amicon Ultra centrifugal filter devices with a 50 000 molecular weight cutoff (Millipore). The concentrations of the purified recombinant proteins were determined by the Bradford assay using bovine serum albumin (BSA) as a protein standard, aliquoted, and stored at −80°C until use.
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