The total proteins extracted from HEp-2 cells were fractionated in a DS-Sepharose column with a BioLogic Duo-Flow system (Bio-Rad). About 40 mg of proteins in 40 mL of 10 mM phosphate buffer (pH 7.4; buffer A) were loaded into the DS-Sepharose column at a rate of 1 mL/min. After loading, the column was washed with 60 mL of buffer A to remove excess and non-binding proteins, followed by eluting with 40 mL of 0.2 M NaCl in buffer A to further remove very weakly binding proteins. Proteins with low to high DS-affinity were eluted with sequential 40-mL step gradients of 0.4 M, 0.6 M, and 1.0 M NaCl in buffer A. Fractions were desalted and concentrated to 0.5 mL with 5-kDa cut-off Vivaspin 20 centrifugal filters (Sartorius). The protein concentration of each fraction was measured. Fractionated proteins were further separated by 1-D SDS PAGE using 4–12% NuPAGE Novex Bis–Tris gels with MES running buffer (Invitrogen). Each gel lane was divided into three sections and subjected to protein sequencing.
Vivaspin 20 centrifugal filters
Manufactured by Sartorius
Sourced in Germany
The Vivaspin 20 is a centrifugal filter device used for the concentration and desalting of biological samples. It features a unique vertical design and a high-performance membrane that allows for efficient separation and recovery of sample components.
Automatically generated - may contain errors
Lab products found in correlation
Mes running buffer, by Thermo Fisher Scientific (1 mentions)
Biologic duoflow system, by Bio-Rad (1 mentions)
Nupage novex bis tris gel, by Thermo Fisher Scientific (1 mentions)
Iron 2 chloride, by Merck Group (1 mentions)
3 aminopropyl trimethoxysilane, by Thermo Fisher Scientific (1 mentions)
Iron 3 chloride, by Merck Group (1 mentions)
Suprapur nitric acid, by Merck Group (1 mentions)
Compass data analysis software, by Bruker (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Hek293f cell, by Thermo Fisher Scientific (1 mentions)
Lipofectamin 2000, by Thermo Fisher Scientific (1 mentions)
Freestyle medium, by Thermo Fisher Scientific (1 mentions)
Escort 4 transfection reagent, by Merck Group (1 mentions)
Methyl mannopyranoside, by Merck Group (1 mentions)
Galanthus nivalis lectin, by Vector Laboratories (1 mentions)
5 protocols using vivaspin 20 centrifugal filters
1
Protein Fractionation and Sequencing
2
Synthesis and Purification of Fluorescent Dye
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Iron (II) chloride, iron (III) chloride, suprapur nitric acid (65%) and suprapur sulphuric acid (96%)were purchased from Merck (purity levels 99%, Darmstadt, Germany). 3-Aminopropyltrimethoxysilane (APTMS) was purchased from Alfa Aesar (MA, USA).
Cardiogreen (indocyanine green) was obtained from Sigma-Aldrich (MO, USA). Vivaspin 20 centrifugal filters (5 kDa MW cut-off) were obtained from Sartorius (Goettingen, Germany). In all experiments, only ultra-pure water was used (18.2 MΩ, Rephile Bioscience and Technology, Shanghai, China).
Cardiogreen (indocyanine green) was obtained from Sigma-Aldrich (MO, USA). Vivaspin 20 centrifugal filters (5 kDa MW cut-off) were obtained from Sartorius (Goettingen, Germany). In all experiments, only ultra-pure water was used (18.2 MΩ, Rephile Bioscience and Technology, Shanghai, China).
3
Mass Spectrometry Analysis of GbpA Protein
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The buffer of GbpA
samples was exchanged to pure water (hydrogenated) by concentration
and subsequent dilution using Vivaspin 20 centrifugal filters (Sartorius).
The protein was stable in water and the samples were concentrated
to 0.3–0.6 mg/mL. Electrospray ionization time-of-flight (ESI-TOF)
mass spectrometry analysis was used for mass determination, using
direct injection into a maXis II ETD HRMS QTOF machine (Bruker). Spectra
containing peaks for differently charged species were obtained and
deconvoluted to a single peak for the +1 species using the charge
deconvolution tool for proteins from the Compass data analysis software
(Bruker). The theoretical and experimental values for the molecular
mass of GbpA are shown inTable S4 . Calculation
of deuteration level for nonlabile hydrogens was performed as described
by Meilleur et al.37 (link) usingeq 1 : where MWdE and MWhE correspond
to the experimentally determined masses of the deuterated and nondeuterated
proteins, respectively, and MWdT and MWhT correspond
to the theoretical masses of both species.
samples was exchanged to pure water (hydrogenated) by concentration
and subsequent dilution using Vivaspin 20 centrifugal filters (Sartorius).
The protein was stable in water and the samples were concentrated
to 0.3–0.6 mg/mL. Electrospray ionization time-of-flight (ESI-TOF)
mass spectrometry analysis was used for mass determination, using
direct injection into a maXis II ETD HRMS QTOF machine (Bruker). Spectra
containing peaks for differently charged species were obtained and
deconvoluted to a single peak for the +1 species using the charge
deconvolution tool for proteins from the Compass data analysis software
(Bruker). The theoretical and experimental values for the molecular
mass of GbpA are shown in
of deuteration level for nonlabile hydrogens was performed as described
by Meilleur et al.37 (link) using
to the experimentally determined masses of the deuterated and nondeuterated
proteins, respectively, and MWdT and MWhT correspond
to the theoretical masses of both species.
4
Production and Purification of SOSIP Trimers
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SOSIPv5.2 trimers were produced as previously described [56 (link)]. In brief, 1 L HEK293F cells (Invitrogen, cat no. R79009) were diluted to a cell density of 1 million cells/ml in FreeStyle Medium (Life Technologies) one hour before transfection. Plasmids encoding for SOSIP and furin were co-transfected in a 4:1 ratio using 1 mg/ml Polyethylenimine Hydrochloride (PEImax) (Polysciences Europe GmBH, Eppelheim, Germany) as the transfection agent. After 6 days, the cells were spun down and the supernatant was filtered using 0.22 μm pore size Steritopes (Millipore, Amsterdam, The Netherlands). The supernatant was run overnight at 4°C (0.5–1.0 ml/min) over a PGT145 affinity column, prepared as previously described [9 (link)]. SOSIP trimers were eluted with 3 M MgCl2 pH 7.2, 20 mM TrisHCl into an equal volume of TN75 buffer (20 mM TrisHCl pH 8.0, 75 mM NaCl). Buffer exchange into TN75 buffer was performed using Vivaspin20 centrifugal filters with 100 kDa MW cut-off (Sartorius, Gӧttingen, Germany).
Viruses were produced by transfection of virus plasmid DNA into HEK293T cells (ATCC, CRL-11268) using lipofectamin2000 (Invitrogen). Supernatant containing virus was harvested 3 days after transfection and used in neutralization assays.
Viruses were produced by transfection of virus plasmid DNA into HEK293T cells (ATCC, CRL-11268) using lipofectamin2000 (Invitrogen). Supernatant containing virus was harvested 3 days after transfection and used in neutralization assays.
5
Production and Purification of HIV-1 Trimeric gp140
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Trimeric gp140 (YU2) was produced as previously described (34 (link)). In brief gp140 was generated by transient transfection of COS7 cells (ATCC) using 5 μg of gp140 plasmid (gift from T.M. Ross, University of Georgia, Athens, GA) and Escort IV Transfection Reagent (Sigma-Aldrich). Purification of gp140 was achieved using a column made with agarose-bound Galanthus nivalis lectin (Vector Laboratories). Gp140 was bound to the column, washed with PBS, and eluted using 1M methyl mannopyranoside (Sigma-Aldrich). The purified protein in eluant was buffer exchanged into sterile PBS and concentrated using Vivaspin 20 centrifugal filters with a 30-kD cutoff (Sartorius). Protein purity was checked by SDS-PAGE. Purified gp140 was stored at −20°C and aliquoted to prevent multiple freeze/thaw cycles.
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