QSOX1 and QSOX1(PDI) variants expressed in bacteria were produced and purified as previously described.11 , 19 For the H72A/P119T mutant produced in mammalian cells, the coding sequence corresponding to Residues 1–546 with an additional six histidines on the carboxy terminus was cloned into the pcDNA3.1 plasmid. The resulting construct was transiently transfected into HEK 293F cells (ThermoFisher). Cells were maintained in FreeStyle 293 medium and transfected using the PEI Max reagent (Polysciences Inc.) with a 1:3 DNA:PEI ratio (w/w). After 5 days, the cell suspension was spun for 10 min at 500 g, the supernatant was removed and spun for 30 min at 9500 g, and the supernatant from the second spin was filtered and purified by nickel‐nitrilotriacetic acid chromatography and size exclusion chromatography.
Pei max reagent
Manufactured by Polysciences
Sourced in United States
PEI MAX reagent is a high-quality transfection reagent used for the delivery of DNA, RNA, and other macromolecules into a variety of cell types. It is a cationic polymer that forms complexes with nucleic acids, facilitating their uptake into cells. PEI MAX is suitable for transient and stable transfection applications.
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Lab products found in correlation
Puromycin, by Fujifilm (3 mentions)
Blasticidin s, by Fujifilm (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Protein g affinity chromatography, by GE Healthcare (2 mentions)
Puromycin, by InvivoGen (2 mentions)
Pspax2, by Addgene (2 mentions)
Mg132, by Peptide Institute (2 mentions)
Uorescence microscopy, by Keyence (2 mentions)
Brefeldin a bfa, by Merck Group (2 mentions)
Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Lenti x 293t cells, by Takara Bio (2 mentions)
Hek293f cell, by Thermo Fisher Scientific (1 mentions)
Hygromycin, by Fujifilm (1 mentions)
Ligation high ver 2, by Takara Bio (1 mentions)
Mg132 mg, by Peptide Institute (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Cos 7, by RIKEN BioResource Center (1 mentions)
S1560, by Biowest (1 mentions)
31 protocols using pei max reagent
1
Recombinant QSOX1 and Variants Purification
2
Lentiviral Stable Cell Line Generation
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Stable cell lines were generated using a lentiviral expression system. HEK293FT cells were transiently cotransfected with lentiviral vectors using PEI MAX reagent (Polysciences, Warrington, PA, USA). Four hours after transfection, the medium was replaced with fresh culture medium. After culturing for 72 h, growth medium containing the lentivirus was collected. HeLa cells were incubated with collected virus-containing medium for 48 h. Uninfected cells were removed using 1 μg/ml puromycin, 5 μg/ml blasticidin S (Wako), or 100 µg/ml hygromycin (Wako).
3
Screening for hCAR Ligands
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Using PEI Max reagent (Polysciences Inc., Warrington, PA), cells were transfected with the appropriate expression and reporter plasmids, and with pGL4.74 (hRluc/TK; Promega, Madison, WI) as an internal standard. After overnight incubation, the cells were treated with individual compounds and solvent control [0.1% dimethyl sulfoxide (DMSO)] for 24 h. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). The firefly luciferase activity was normalized against that of Renilla luciferase. Primary screening for hCAR ligands was conducted using a single luciferase reporter assay as follows: cells were transfected with pcDNA-GAL4-hCAR/LBD or pcDNA-GAL4-hCAR/LBD(+3a.a.) and pG5luc reporter plasmids.
4
Lentiviral CRISPR/Cas9 Transduction Protocol
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Lentiviral transfer vector was co-transfected with standard packaging plasmids into 293T cells using PEI MAX reagent (Polysciences) and the supernatant fluids harvested at 72 h were filtered through a 0.22 μm syringe filter. Production of sgRNA CRISPR/Cas9 lentivirus was similarly carried out by co-transfecting 293T cells with sgRNA expressing vectors listed in Supplementary Table S4 . Lentivirus transduction was performed by supplementation of 8 μg/ml polybrene, followed by antibiotic selection with 6 μg/ml puromycin. Antibiotic-resistant bulk cell populations were used for experiments to avoid clonal biases.
5
High-Throughput Antibody Variant Expression
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The coding sequences for variable domains of antibody 492.1 were fused to human antibody constant regions [72 (link)]. Mutations were introduced by site-directed mutagenesis into the resulting hybrid antibody expression plasmids according to published procedures [73 (link)]. Plasmids were transfected into suspension-adapted suspension-HEK 293F cells. The day before transfection, cells were split to 0.7 x 106 cells/ml. For parallel expression of the parent hybrid antibody and the 20 variants, transfections were performed using 0.5 μg of each plasmid (heavy and light Ab chains) mixed with 3 μg PEI Max reagent (Polysciences Inc.) and incubated 20 min in 24-well tissue culture trays prior to addition of 1 ml cells per well. Plates were then agitated vigorously in a tissue culture incubator/shaker to prevent cell settling. After 4 days, cultures were transferred to microfuge tubes, and cells were pelleted by centrifugation at 500 x g for 10 min. Supernatants were transferred to fresh microfuge tubes, from which aliquots were taken for quantification of antibody expression and activity. For purification of selected Ab designs, transfections were done in 40 ml volumes, and plasmid and PEI Max amounts were scaled up accordingly. Cultures were grown for 6 days, and Ab was purified from the supernatant by protein G affinity chromatography (GE Healthcare).
6
Lentiviral Knockdown of PRRX1 in 143B Cells
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pLKO.1puro, a lentiviral vector plasmid purchased from Addgene (#8453; Watertown, MA, USA), was digested with AgeI and EcoRI and then ligated with annealed primers using Ligation High ver 2 (Takara Bio). The following primers were used: shPRRX1#1 forward 5ʹ-CCG GTG ACA TTT AGG GTA TAA AGA TCT TCA AGA GAG ATC TTT ATA CCC TAA ATG TCT TTT TTG-3ʹ, shPRRX1#1 reverse 5ʹ-AAT TCA AAA AAG ACA TTT AGG GTA TAA AGA TCT CTC TTG AAG ATC TTT ATA CCC TAA ATG TCA-3ʹ; shPRRX1#2 forward 5ʹ-CCG GTG CAG CGA AGG AAT AGG ACA ACT TCA AGA GAG TTG TCC TAT TCC TTC GCT TTT TTG-3ʹ, shPRRX1#2 reverse 5ʹ-AAT TCA AAA AAG CAG CGA AGG AAT AGG ACA ACT CTC TTG AAG TTG TCC TAT TCC TTC GCT GCA-3ʹ. For lentiviral production, pLKO.1 puro constructs were transfected together with packaging vectors, including pMDLg/pRRE, pRSV-Rev, and pMD2.G, to lentiX293T cells using PEI-MAX reagent (Polysciences, Warrington, PA, USA). Culture media were replaced with fresh media 12 h after transfection. Cells were cultured for 48 h, and culture supernatants containing lentivirus were passed through a 0.45 µM PVDF filter (Hawach Scientific, Xi'an, China). Lentivirus solutions were stored at −80 °C until use. For lentiviral infection, 143B cells were treated with the lentiviral solution for 24 h. After culturing for another 24 h without lentivirus, cells were treated with 1 µg/mL puromycin (Wako) to select lentivirus-infected cells.
7
Establishment of CRELD2-Deficient Neuro2a Cells
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Neuro2a cells were maintained in Dulbecco’s Modified Eagle’s Minimum Essential Medium containing 5% fetal bovine serum. Transfection of the indicated constructs was performed using the PEI-MAX reagent (Polysciences) as previously described5 (link),17 (link). For the establishment of CRELD2-deficient cells, Neuro2a cells transfected with the indicated constructs; the gRNA, hCas9 and donor genes, were cultured with hygromycin, and the resultant cells were used in this study17 (link). During these selections, the parental wild-type (WT) Neuro2a cells were maintained with the normal culture medium and were used as control cells for the following experiments. A CRELD2-deficient single clone was obtained after the seeding and growth of a cell in 96-well plate. In each experiment, parental and each deficient cell were seeded into 96- or 12-well plates or 3.5-cm dishes with non–hygromycin containing culture medium. After that, the cells were treated with or without thapsigargin (Tg, 0.1 μM), tunicamycin (Tm, 1 μg/ml), breferdin A (BFA, 2.5 μg/ml), cycloheximide (CHX, 10 μg/ml) (Sigma-Aldrich), MG132 (MG, 10 μM) (Peptide Institute) or serum-deprived medium (serum free, SF) for the indicated time period.
8
Transient Transfection of HEK293T and COS-7 Cells
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HEK293T (Tiscornia et al., 2006 (link)) and COS-7 cells (RCB0539, RIKEN BioResource Research Center) were cultured in DMEM (11995–065, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (S1560, Biowest) and 1% penicillin-streptomycin-glutamine (10378–016, Thermo Fisher Scientific) at 37°C under 5% CO2. For HEK293T cells, plasmid DNA were transiently transfected (Tiscornia et al., 2006 (link)) and cultured for 48 h. For COS-7 cells, PEI MAX reagent (Polyscience) was used for transient transfection, followed by culturing for 24 h.
9
Immunoprecipitation of BMAL1 and CLOCK
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HEK293A cells were transfected with pcDNA3.0-BMAL1-His, PcDNA3.0-6XMyc-CLOCK or PcDNA3.0-6XMyc-CLOCKC267A using Polyethylenimine PEI MAX reagent (Polysciences 247,651). Twenty-four hours after transient transfection, cells were treated with the indicated compounds for 8 h and homogenized in lysis buffer (50 mM Tris pH7.5, 150 mM NaCl 0.5% Triton X-100, 5% glycerol and protease inhibitor). Immunoprecipitation was performed overnight using Anti-c-Myc Agarose beads (Thermo Fisher 20168). Beads were washed three times with lysis buffer. The resultant protein samples were resolved by SDS-PAGE and immunoblotted with antibodies that are described in Supplemental Table 3.
10
Purification of FLAG-tagged IRF1 Proteins
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FLAG-tagged IRF1 proteins were ectopically expressed in 293FT cells grown on 15 cm dishes using PEI MAX reagent (Polysciences). Cells transfected with empty vector were processed in parallel. Twenty-four hours after transfection, the cells were scraped into a lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 50 mM sodium fluoride, 1 mM Na3VO4 and 1% Triton X-100) supplemented with a Complete protease inhibitor cocktail (Roche). Clarified lysates were subsequently purified by binding to Anti-FLAG M2 affinity agarose gel (Sigma), followed by elution with the FLAG peptide (0.2 mg/ml). The resulting eluate was concentrated using an Amicon Ultra 10K Centrifugal filters (Millipore) and diluted in wash buffer (Tris-buffered saline containing 1% Nonidet P-40). This procedure was repeated five times to reduce the concentration of the FLAG peptide.
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