Subconfluent BHK cells (ca. 3.0 × 106) in one well of a six-well culture plate was transfected with 2.0 μg/well of the pcDL-SRα expression vector encoding each protein. After 12 h or 24 h of incubation at 37°C, the cells were treated with 0.3 mg/ml of Sulfo-NHS-LC-Biotin (Thermo Scientific) in PBS supplemented with 0.1 mM CaCl2 and 1 mM MgCl2 at 23°C for 30 min and lysed on ice with 500 μl/well of lysis buffer: Sulfo-NHS-LC-Biotin is a membrane impermeable reagent that biotinylates the primary amines on the cell surface (Staros, 1982 (link)). The biotinylated cell-surface proteins in 150 μl of the cell lysates were then immunoprecipitated with monoclonal antibodies specific for either the HPIV2 HN protein (MAb 173-1A) or the SV41HN protein (MAb 127A-1), or with the rabbit antiserum specific for PIV5 F2. The biotinylated and immuneprecipitated proteins were subjected to SDS-PAGE under reducing or non-reducing conditions and the separated proteins were electroblotted to nitrocellulose membrane, treated with streptavidin-biotin-peroxidase complex, and detected by ECL as described above. The intensity of the HN or the F protein band was quantified with the aid of a graphics software, NIH ImageJ ver.1.45s, and relative surface localization level was estimated.
Sulfo nhs lc biotin
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
Sulfo-NHS-LC-Biotin is a water-soluble, amine-reactive biotinylation reagent. It is used for the covalent modification of proteins and other molecules containing primary amino groups.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin agarose bead, by Thermo Fisher Scientific (17 mentions)
Neutravidin agarose beads, by Thermo Fisher Scientific (6 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (6 mentions)
Nitrocellulose membrane, by GE Healthcare (5 mentions)
Lysis buffer, by Thermo Fisher Scientific (5 mentions)
Ecl detection system, by GE Healthcare (5 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (5 mentions)
Neutravidin beads, by Thermo Fisher Scientific (4 mentions)
Streptavidin beads, by Thermo Fisher Scientific (4 mentions)
Neutravidin agarose resin, by Thermo Fisher Scientific (4 mentions)
Fluorescent neutravidin beads, by Thermo Fisher Scientific (3 mentions)
Streptavidin agarose beads, by Merck Group (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Dulbecco phosphate buffered saline (dpbs), by Merck Group (3 mentions)
1 μm fluorescent neutravidin beads, by Thermo Fisher Scientific (3 mentions)
Lsr 2, by BD (3 mentions)
Complete protease inhibitor cocktail, by Roche (3 mentions)
Magnetic column, by Miltenyi Biotec (2 mentions)
Beckman j 6b centrifuge, by Beckman Coulter (2 mentions)
Streptavidin conjugated magnetic microbeads, by Miltenyi Biotec (2 mentions)
163 protocols using sulfo nhs lc biotin
1
Quantifying Cell Surface Protein Localization
2
Biotinylation and Purification of NMDAR
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Five 100 mm Petri dishes with HEK293 cells expressing the different NMDAR constructs were washed three times with ice-cold PBS, pH 8.0, to remove any contaminating proteins.
Cells were suspended at a concentration of ~10 million cells/ml in PBS, pH 8.0. 1 mg of sulfo-NHS-LC-Biotin (Thermo Scientific) per ml of reaction volume was added to the cells and incubated at 4 °C for 30 min. After this, cells were washed three times with ice-cold PBS and 100 mM glycine to quench any remaining biotinylation reagent. The cell surface proteins are now biotinylated on exposed lysine residues.
Biotinylated proteins were purified using the Thermo Scientific Pierce Cell Surface Protein Isolation Kit® following manufactured instructions. For loading control of western blots, an aliquot of total protein (before purification of biotinylated protein) was immunoprecipitated using anti-GFP selective polyclonal antibody (Thermo Scientific, A-11122), and the immunoprecipitation kit (Abcam ab206996) following the manufacturer instructions (both NMDAR subunits had GFP fused). For the identification of the NMDAR on the western blots, the selective rabbit monoclonal antibody (ab109182) was used for NR1 and antibody (ab133265) for NR2A (Abcam).
Cells were suspended at a concentration of ~10 million cells/ml in PBS, pH 8.0. 1 mg of sulfo-NHS-LC-Biotin (Thermo Scientific) per ml of reaction volume was added to the cells and incubated at 4 °C for 30 min. After this, cells were washed three times with ice-cold PBS and 100 mM glycine to quench any remaining biotinylation reagent. The cell surface proteins are now biotinylated on exposed lysine residues.
Biotinylated proteins were purified using the Thermo Scientific Pierce Cell Surface Protein Isolation Kit® following manufactured instructions. For loading control of western blots, an aliquot of total protein (before purification of biotinylated protein) was immunoprecipitated using anti-GFP selective polyclonal antibody (Thermo Scientific, A-11122), and the immunoprecipitation kit (Abcam ab206996) following the manufacturer instructions (both NMDAR subunits had GFP fused). For the identification of the NMDAR on the western blots, the selective rabbit monoclonal antibody (ab109182) was used for NR1 and antibody (ab133265) for NR2A (Abcam).
3
Aspergillus fumigatus Conidia Labeling and Infection
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A. fumigatus CEA10 (46 (link)) and CEA10-RFP (47 ) strains were cultured on glucose minimal medium slants at 37 °C for 4–7 days prior to harvesting conidia for experimental use. To generate AF633-labeled or FLARE conidia for experimental use, 7×108 CEA10 (for AF633-labeled) or CEA10-RFP (for FLARE) conidia were rotated in 10 μg/ml Sulfo-NHS-LC-Biotin (Thermo Scientific) in 1 ml of 50 mM carbonate buffer (pH 8.3) for 2 hr at 4 °C, incubated with 20 μg/ml Streptavidin, Alexa Fluor™ 633 conjugate (Molecular Probes) at 37 °C for 1 h, resuspended in PBS and 0.025% Tween 20 for use within 24 hr (12 (link)).
To generate morphologically uniform heat-killed swollen conidia, 5×106/ml conidia were incubated at 37 °C for 14 hours in RPMI-1640 and 0.5 μg/ml voriconazole and heat-killed at 100 °C for 30 minutes (48 (link)). To infect mice with 3–6×107A. fumigatus cells, conidia were resuspended in PBS, 0.025% Tween-20 at a concentration of 0.6–1.2×109 cells/mL and 50 µl of cell suspension was administered via the intratracheal route to mice anesthetized by isoflurane inhalation.
To generate morphologically uniform heat-killed swollen conidia, 5×106/ml conidia were incubated at 37 °C for 14 hours in RPMI-1640 and 0.5 μg/ml voriconazole and heat-killed at 100 °C for 30 minutes (48 (link)). To infect mice with 3–6×107A. fumigatus cells, conidia were resuspended in PBS, 0.025% Tween-20 at a concentration of 0.6–1.2×109 cells/mL and 50 µl of cell suspension was administered via the intratracheal route to mice anesthetized by isoflurane inhalation.
4
Preparation and Biotin-Labeling of Synthetic Aβ
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Synthetic Aβ40 monomers and Aβ42 protofibrils were prepared as previously described [18, 40, 41 (link)]. Synthetic Aβ40 (Polypeptide Laboratories AB, Limhamn, Sweden) dissolved in 10 mM NaOH, was diluted in 2×phosphate buffered saline (PBS) to 100μM. Synthetic Aβ42 (American Peptide Company Inc., Sunnyvale, CA, USA) dissolved in 10 mM NaOH, diluted in 10×PBS to 443μM (2 mg/ml), was incubated for 30 min at 37°C and centrifuged for 5 min at 17 900×g to remove any insoluble aggregates and then diluted with PBS to a final concentration of 100μM. Both Aβ40 monomers and Aβ42 protofibrils were biotinylated with Sulfo-NHS-LC-Biotin (Thermo Fischer Scientific Inc., Waltham, MA, USA) according to manufacturer’s guidelines with an Aβ/biotin molar ratio of 1/20. The Aβ42 protofibril concentration is expressed in monomer concentration units.
5
Surface Biotinylation of Cardiomyocytes
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Surface-protein biotinylation was measured as previously described [25 (link)] with the modifications described below. After culturing with high palmitate medium or the transfection with AdPKD, aRCMs were incubated for 30 min with (or without) 100 nm insulin. Subsequently the cells were biotinylated with the cell-impermeable reagent sulfo-NHS-LC-biotin (0.5 mg/mL dissolved in M199 medium, Thermo Fisher Scientific, Fremont, CA, USA) for 5 min at 37 °C. The rest steps of surface-protein biotinylation assay were carried out as previously described [25 (link)]. Insulin-regulated aminopeptidase protein (IRAP, which reflects GLUT4 trafficking) was detected by Western blotting.
6
Virus Immobilization Methods
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Unless otherwise specified, viruses were immobilized using poly-L-Lysine. Coverslips (25x65mm, thickness number 1, VWR) were heated in a furnace at 500°C for 1 hour to burn off any dust. A silicone gasket (Grace Bio-Labs, USA) was added to the glass slide and 0.01% poly-L-Lysine (Sigma) solution was added to the wells for 30 minutes. The excess was removed and the chambers were washed three times with MilliQ water and the slide allowed to dry. As an alternative chitosan was used in place of poly-L-Lysine (0.015% chitosan powder (Sigma) in 0.1M ethanoic acid).
For specific immobilisation of viruses via biotinylation, passivated microscope slides were prepared by washing in acetone and Vectabond solution (Vector Laboratories) before being incubated with NHS-PEG:Biotin-NHS-PEG in an 80:1 ratio. 0.5 mg/mL neutravidin was incubated for 10 minutes at room temperature on the slide shortly before virus was added. Viruses were biotinylated by incubation in a 1 mg/mL Sulfo-NHS-LC-Biotin (ThermoFisher) for 3 hours at 37°C before being fixed and immunolabelled as described below.
For specific immobilisation of viruses via biotinylation, passivated microscope slides were prepared by washing in acetone and Vectabond solution (Vector Laboratories) before being incubated with NHS-PEG:Biotin-NHS-PEG in an 80:1 ratio. 0.5 mg/mL neutravidin was incubated for 10 minutes at room temperature on the slide shortly before virus was added. Viruses were biotinylated by incubation in a 1 mg/mL Sulfo-NHS-LC-Biotin (ThermoFisher) for 3 hours at 37°C before being fixed and immunolabelled as described below.
7
Surface Biotinylation and NeutrAvidin Pulldown
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The surface biotinylation and NeutrAvidin pull down methods have been described previously (Shafer et al., 2011 (link); Onishi et al., 2013 (link)). Briefly, 48 hr after transfection with indicated plasmids, HEK293T cells (seeded on 20 μg/ml PDL-coated six-well plate) cells were washed with ice-cold PBS (pH 8.0) three times and incubated with 1 mg/ml Sulfo-NHS-LC-Biotin (ThermoFisher Scientific)/PBS for 2 min at room temperature to initiate the reaction, followed by incubation on ice for 1 hr. After quenching active biotin by washing with ice-cold 100 mM Glycine/PBS twice followed by normal ice-cold PBS, the cell lysates were incubated with NeutrAvidin agarose for 2 hr and then precipitated. For quantification, three independent experiments were performed and the band intensity was quantified with ImageJ. Statistical analysis was performed with Prism7 (GraphPad Software).
8
Biotinylation of HIV gp120 and Influenza HA
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HIV gp120 (strain YU2) antigen (Immune Technology, IT-001-0027p YU-2) was biotinylated at lysine residues using sulfo-NHS-LC-biotin (Thermo Scientific, 21,935) according to the manufacturer's instructions. As an alternative antigen, influenza hemagglutinin (HA) (California H1N1 2009, Immunetech, IT-003-SW12ΔTMp) was used and biotinylated in the same way. To remove residual biotin after the reaction, antigens were buffer exchanged into phosphate buffered saline (PBS; Sigma-Aldrich, Saint Louis, USA) with Zeba Spin Desalting Columns (Thermo Scientific, 87766) with 40K MWCO according to the manufacturer's instructions. The biotinylated antigens were incubated, individually, with red 1.0 μm fluorescent neutravidin beads (Thermo Fisher, F8775) at 37 °C in a low-binding microcentrifuge tube (Corning, CLS3207). Beads were subsequently washed twice in 5% PBS- BSA, and then resuspended at a final dilution of 1:100 in 0.1% PBS-BSA. Antigen-coated beads were stored for up to 2 days at 4 °C and protected from light.
9
Internalization of Cellular Prion Protein
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L929 cells were plated and transiently transfected with siRNA as described previously. At 72 h post transfection, cells were rinsed with ice cold PBS and biotinylated with 250 mg/ml Sulfo-NHS-LC-Biotin (Thermo Scientific, Schwerte, Germany) on ice for 20 min. Cells were subsequently washed with cold PBS, incubated with 20 mM Glycin/50 mM NH4Cl on ice to quench the biotinylation reaction and rinsed again with ice cold PBS. Cultures were chased at 37 °C for 60 min to allow internalization of PrPC. Cells were subsequently incubated with or without trypsin on ice for 10 min. Proteolysis was terminated by addition of soybean trypsin inhibitor. After lysis of cells, PrPC was immunoprecipitated using the monoclonal antibody 4H11 at 4 °C over night and subjected to SDS-PAGE followed by immunoblot. Biotinylated PrPC was detected using horseradish peroxidase- (HRP-) conjugated streptavidin and signals were analyzed densitometrically. The amount of internalized PrPC (+Trypsin) was expressed as a percentage of the amount of total biotinlytated PrPC detected in control cells without Trypsin treatment (−Trypsin). The mean signal value in this sample group was set to 100%.
10
Isolation of Young and Old Cryptococcus Cells
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Wt or mutant C. neoformans cells were grown in YPD medium and isolated at 0–2 or 10-generation-old as described previously (Bouklas et al., 2013 (link)). Briefly, newly budded C. neoformans cells were isolated by elutriation (Beckman JE-5.0 rotor in a Beckman J-6B centrifuge; Beckman Instruments, Inc.) and labeled with Sulfo-NHS-LC-Biotin (Thermo Scientific). The newly budded and labeled cells were grown for several generations (10 generations), and collected by first binding them to streptavidin-conjugated magnetic microbeads (Miltenyi Biotec), then isolating them on a magnetic column (Miltenyi Biotec). Unbound young yeast cells (0-2 generations old) that washed off the column and had been exposed to similar manipulations were used as controls. Purity of old cells was confirmed by fluorescein isothiocyanate (FITC)-staining of the streptavidin-labeled cells.
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