Monolayers of 17Cl1 cells grown in Eagle's medium containing 10% fetal bovine serum were inoculated at a multiplicity of 1 PFU/cell. Infections were allowed to proceed for 12–16 h, to a point where syncytia formation was maximal but little or no cell lysis or detachment had occurred. Released virus in harvested extra-cellular medium was precipitated with polyethylene glycol and resuspended in magnesium- and calcium-free phosphate-buffered saline, pH 7.4 (PBS). Virions were sedimented onto cushions of 60% sucrose in PBS by centrifugation at 151,000 × g for 2.5 h in a Beckman SW41 rotor at 4 °C. Samples were removed from cushions, diluted with PBS to contain 10% sucrose and layered onto 10–20–40–60% sucrose step gradients. Virion bands were collected from the 20–40% sucrose interface after centrifugation at 151,000 × g for 2.5 h in a Beckman SW41 rotor at 4 °C. For the SARS-CoV M chimera set (Fig. 2 ) and the domain N3 mutant set (Fig. 3 ), virions were further purified by pulldown with anti-M monoclonal antibody J.1.3 and nProtein A Sepharose beads (GE Healthcare) exactly as described previously (Kuo and Masters, 2013 (link)).
Nprotein a sepharose beads
Manufactured by GE Healthcare
NProtein A Sepharose beads are a type of chromatography resin used for the purification of antibodies and other proteins that interact with Protein A. The beads are composed of Sepharose, a cross-linked agarose matrix, with Protein A covalently coupled to the surface. This allows for the selective capture and separation of Protein A-binding proteins from complex mixtures, such as cell culture supernatants or tissue extracts.
Automatically generated - may contain errors
Lab products found in correlation
Speedvac system, by Thermo Fisher Scientific (2 mentions)
Ab5070, by Abcam (2 mentions)
Anti psi, by Biomatik (2 mentions)
Sw41 rotor, by Beckman Coulter (1 mentions)
Easy nlc 1000 system, by Thermo Fisher Scientific (1 mentions)
Speedvac, by Thermo Fisher Scientific (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Flag antibody, by Merck Group (1 mentions)
Anti psi antibody, by Biomatik (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
8 protocols using nprotein a sepharose beads
1
SARS-CoV Virion Purification and Characterization
2
Affinity Enrichment and Mass Spectrometry Analysis of Tryptic Peptides
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We began by conjugating pan anti-Kmea antibodies to nProtein A Sepharose beads (GE Healthcare Bio-Sciences Corp., Piscataway, NJ). The bead conjugated antibodies were then incubated in the presence of tryptically digested peptides at 4 °C with gentle agitation. We washed the beads with NETN buffer (50 mM Tris-Cl pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.5% NP-40) three times. This was followed by two washes in ETN buffer which lacked the 0.5% NP- 40. This was followed once with deionized water. We then eluted peptides using 0.1% trifluoroacetic acid. The eluted peptides were then dried in a SpeedVac (Thermo Fisher Scientific Inc.).
Dried peptides samples were dissolved in HPLC Buffer A (0.1% formic acid in water, v/v) and loaded into a homemade capillary column (10-cm length × 75-mm ID, 3-μm particle size, Dr. Maisch GmbH, Ammerbuch, Germany) attached to an EASY-nLC 1000 system (Thermo Fisher Scientific Inc., Waltham, MA). We separated and eluted peptides along a gradient of 2 to 90% HPLC Buffer B (0.1% formic acid in acetonitrile, v/v) in HPLC Buffer A at a flow rate of 200 nL min−1 over 60 min. The peptides were ionized and analyzed using a Q-Exactive mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA).
Dried peptides samples were dissolved in HPLC Buffer A (0.1% formic acid in water, v/v) and loaded into a homemade capillary column (10-cm length × 75-mm ID, 3-μm particle size, Dr. Maisch GmbH, Ammerbuch, Germany) attached to an EASY-nLC 1000 system (Thermo Fisher Scientific Inc., Waltham, MA). We separated and eluted peptides along a gradient of 2 to 90% HPLC Buffer B (0.1% formic acid in acetonitrile, v/v) in HPLC Buffer A at a flow rate of 200 nL min−1 over 60 min. The peptides were ionized and analyzed using a Q-Exactive mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA).
3
Histone Extraction and Peptide Enrichment
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Histones from human MCF-7 or mouse BMDM cells were extracted using a standard acid extraction protocol19 (link), and subjected to trypsin digestion as per the manufacturer’s instructions. Pan anti-Kla or pan anti-Kac antibodies were first conjugated to nProtein A Sepharose beads (GE Healthcare BioSciences, Pittsburgh, PA) and then incubated with tryptically digested histone peptides with gentle agitation overnight at 4 °C. The beads were then washed three times with NETN buffer (50 mM Tris-Cl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40), two times with ETN buffer (50 mM Tris-Cl pH 8.0, 100 mM NaCl, 1 mM EDTA) and once with water. Peptides were eluted from the beads with 0.1% TFA and dried in a SpeedVac system (Thermo Fisher Scientific, Waltham, MA).
4
Histone Extraction and Peptide Enrichment
5
Chromatin Immunoprecipitation Protocol
6
Co-immunoprecipitation of AGO1 and Psi
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Co-IP was performed using 25 wild-type third instar larval heads dissociated in cold lysis buffer (50 mM Tris pH 7.5, 1.5 mM MgCl2, 125 mM NaCl, 0.2% NP40, 5% glycerol, 1× protease inhibitor cocktail). Following homogenisation, protein was collected by centrifugation at 13,000 g for 10 min at 4°C. The extract was pre-cleared by incubation with nProtein A Sepharose beads (GE Healthcare Life Science) for 1 h at 4°C with rotation and the supernatant collected by centrifugation at 13,000 g . Equal amounts of pre-cleared protein lysate were incubated with either anti-AGO1 antibody (Abcam, ab5070, 1:70), anti-Psi antibody (custom generated rabbit polyclonal antibody, Biomatik, 1:100) or without antibody (mock IP control) overnight at 4°C. Beads were washed five times with lysis buffer and the eluent resolved using 10% SDS PAGE/western blot with appropriate primary antibody before detection with Li-Cor Odyssey IR.
7
Drosophila Co-Immunoprecipitation Protocol
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Co-IP for Drosophila was performed using 25 wild type 3rd instar larval heads dissociated in cold lysis buffer (50 mM Tris pH 7.5, 1.5 mM MgCl2, 125 mM NaCl, 0.2% NP40, 5% glycerol, 1x Protease inhibitor cocktail, Roche). Following homogenization, protein was collected by centrifugation at 12 000 rpm for 10 min at 4°C. The extract was pre-cleared by incubation with nProtein A Sepharose TM beads (GE Healthcare Life Science) for 1 h at 4°C with rotation and the supernatant collected by centrifugation at 12 000 rpm. Equal amounts of pre-cleared protein lysate were incubated with either guinea pig anti-MED17 (Gift from Michael Marr), mouse anti-Cdk8 (Abcam, ab52779) or anti-Psi (custom rabbit polyclonal antibody, Biomatik) antibodies overnight at 4°C. Beads were washed with lysis buffer 5 times and the eluent resolved using 10% SDS-PAGE/Western with anti-Psi antibody (1/1000) prior to detection with Li-Cor Odyssey IR detection.
8
Co-immunoprecipitation of AGO1 and Psi Proteins
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Co-Immunoprecipitation (Co-IP) was performed using 25 wild type 3rd instar larval heads dissociated in cold lysis buffer (50 mM Tris pH 7.5, 1.5 mM MgCl2, 125 mM NaCl, 0.2% NP40, 5% glycerol, 1x Protease inhibitor cocktail). Following homogenization, protein was collected by centrifugation at 12 000 rpm for 10 min at 4°C. The extract was pre-cleared by incubation with nProtein A Sepharose TM beads (GE Healthcare Life Science) for 1 hour at 4°C with rotation and the supernatant collected by centrifugation at 12 000 rpm. Equal amounts of pre-cleared protein lysate were incubated with either anti-AGO1 (Abcam, ab5070) or anti-Psi (custom generated rabbit polyclonal antibody, Biomatik) antibodies overnight at 4°C. Beads were washed with lysis buffer 5 times, and the eluent resolved using 10% SDS PAGE/Western with appropriate primary antibody prior to detection with Li-Cor Odyssey IR detection.
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