Anti-biotin antibody 1 (Abcam, no. ab53494), anti-biotin antibody 2 (Bethyl Laboratories, no. 150-109A), streptavidin-HRP (Abcam, no. ab7403), protein G beads (EMD Millipore, no. 16-266), high-capacity NeutrAvidin agarose (Thermo Fisher Scientific, no. 29202), biotin (Sigma-Aldrich, no. B4501), biotin-2′,2′,3′,3′-d4 (Sigma-Aldrich, no. 809068), Lipofectamine 2000 (Thermo Fisher Scientific, no. 11668019), biotin-phenol (Iris Biotech, no. CDX-B0270-M100), sequencing-grade trypsin (Promega, no. V5113), GalT1 enzymatic labeling kit (Invitrogen, no. C33368), PNGase F (New England Biolabs, no. P0704S), Click-IT Biotin DIBO Alkyne (Thermo Fisher Scientific, no. C10412), and trypsin (Worthington Biochemical Corporation, no. LS003741) were used.
High capacity neutravidin agarose
Manufactured by Thermo Fisher Scientific
High Capacity Neutravidin Agarose is a type of affinity chromatography media. It consists of Neutravidin, a modified version of avidin, immobilized on an agarose support. This media is designed to bind and capture biotinylated molecules.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Sequencing grade trypsin, by Promega (1 mentions)
Streptavidin hrp, by Abcam (1 mentions)
Protein g beads, by Merck Group (1 mentions)
Trypsin, by Worthington (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
Biotin phenol, by Iris Biotech (1 mentions)
Sulfo nhs ss biotin, by Apexbio (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Ni nta agarose bead, by Qiagen (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Dntps, by Takara Bio (1 mentions)
10k mwco, by Thermo Fisher Scientific (1 mentions)
Taq polymerase, by Takara Bio (1 mentions)
Hydrogen peroxide h2o2, by Sinopharm (1 mentions)
Mini dialysis devices, by Thermo Fisher Scientific (1 mentions)
Oil red o, by Merck Group (1 mentions)
Hematoxylin, by Beyotime (1 mentions)
Pierce cell surface protein isolation kit, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
9 protocols using high capacity neutravidin agarose
1
Comprehensive Protein Biotinylation Methods
2
Biotinylation and Enrichment of HCN1 Channels
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HEK293 cells were briefly washed with cold PBS prior to incubation with 2 mg/mL Sulfo-NHS-SS-Biotin (APExBIO cat. # A8005) in cold PBS for thirty minutes on ice. The reaction was quenched by the addition 50 mM Tris-HCl, 150 mM NaCl, pH 7.5). Cells were briefly washed with PBS prior to addition of lysis buffer. For each replicate, equal amounts of protein (200–300 μg) for each experimental sample was added to 75 μL of High capacity Neutravidin agarose (ThermoFischer Scientific cat. # 29202) and incubated at 4°C overnight. Resin was washed with lysis buffer supplemented with 0.5% SDS. Protein was eluted by incubation for 5 minutes at 75°C in 2X reducing sample buffer prepared from 4X LDS sample buffer and 10X reducing agent. Eluted protein was then examined by western blot. T-test was used to compare mRuby3-HCN1 and mRuby3-HCN1Δ3, 14-3-3. 1-way ANOVA followed by Dunnett’s multiple comparison was used to compare SNAP-HCN1Δ804–814; Δ861–871 and SNAP-HCN1S810A; S867A to SNAP-HCN1. All data presented as mean ± standard deviation.
3
Mannosyl Acceptor Peptide Pulldown
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Standard reactions were performed as described above. Instead of GST-αDG, synthetic peptides (200 μm ) carrying a C-terminal Biotin tag were included as mannosyl acceptor. After stop of the reaction, mixtures were centrifuged for 10 min at 20,000 × g at 4 °C. The supernatant was incubated with 20 μl of slurry of High Capacity Neutravidin®-agarose (Thermo Scientific) for 1 h at 4 °C. Beads were washed two times with PBS plus 1% Triton X-100 and two times with PBS. Incorporated radioactivity was measured by liquid scintillation counting.
4
Protein Isolation and Analysis Techniques
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We obtained cycloheximide, diethylene triamine pentacetate acid (DTPA), N-ethylmaleimide (NEM), iodoacetamide, catalase, palmitic acid, fatty acid-free bovine serum albumin (BSA), protein inhibitors, 2′, 7′-Dichlorofluorescein diacetate (DCFH-DA) fluorescent dyes, Oil Red O, anti-Myc-coupled agarose beads from Sigma; high capacity NeutrAvidin agarose, mini dialysis devices, 10K MWCO, and ECL from ThermoFisher Scientific. We obtained Ni-NTA agarose beads from Qiagen; MG132 from Calbiochem; M-MLV Reverse Transcriptase from Promega; KOD Hot Start DNA Polymerase from Novagen; Taq polymerase, dNTPs, and X-Gal from TaKaRa; Quick change Site-Directed Mutagenesis kit from Stratagene; Cysteine Sulfenic acid probe DCP-Bio1 from Millipore; hydrogen peroxide (H2O2) from Sinopharm Chemical Reagent Co.,Ltd.; hematoxylin from Beyotime.
5
Affinity Purification of Sulfo-NHS-SS-Biotin Labeled Peptides
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Sulfo-NHS-SS-biotin labelled peptides were immobilized on high capacity neutravidin agarose (Thermo Scientific). The binding efficiencies of the column were examined by dot-blot experiments (Supplementary Methods), in order to determine the optimal volume of neutravidin agarose (100 µl/sample). Labelled mixtures were incubated on affinity columns for 1 hour at room temperature. Columns were washed extensively to reduce the number of non-specific peptides or contaminants as described by Lango et al.43 (link). The covalently modified peptides were eluted from the column with 50 mM DTT in 50 mM NH4HCO3 (pH = 8) for 1 hour at 37 °C, which were followed by an alkylation step with 110 mM iodoacetamide in dark at room temperature.
6
Biotin-Poly(I:C) Pulldown of DHX16 Protein
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500 ng of biotin-labeled Poly(I:C) (HMW) (InvivoGen) was incubated with High Capacity NeutrAvidin Agarose (Thermo Scientific) in NT2 buffer (50 mM Tris-HCL pH 7.4, 150 mM NaCl, 1 mM MgCl2, and 0.05% (v/v) IGEPAL CA-630) overnight at 4°C on a rotating platform. RNA-bound beads were washed seven times in NT2 buffer and incubated further with purified DHX16 proteins in NT2 buffer overnight at 4°C on a rotating platform. Bound complexes were washed seven times in NT2 buffer and eluted by heating samples in 2X Laemmli buffer containing 2-Mercaptoethanol for 10 min at 95°C.
7
Cell Surface Biotinylation Isolation
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Cell-surface biotinylation was carried out as previously described [5 (link)] using the Pierce cell-surface protein isolation kit (Thermo Scientific) as per the manufacturer’s instructions. Briefly, cells were labelled with sulfosuccinimidyl-2-[biotinamido] ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-Biotin) in PBS (Mg/Ca) for 30 min on ice, the unreacted biotin was quenched with 100 mM glycine and protein content in cells assayed by BCA after lysis. To precipitate biotinylated surface proteins, equal protein amounts from cell lysates were incubated with High Capacity Neutravidin Agarose (Thermo Scientific). Non-bound ‘intracellular’ proteins were separated before agarose was washed with ice-cold ‘high salt’ wash buffer (500 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl pH 7.5) and ice-cold ‘no salt’ wash buffer (10 mM Tris-HCl pH 7.5). Bound proteins were eluted with SDS-PAGE sample buffer containing 50 mM DTT, before analysis by western blotting.
8
CD44 Phosphorylation by CK2α Assay
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Either human or murine CD44-Fc-AviTag N-terminal domain protein (1 mg/mL) was incubated in the presence of CK2α, or mutant, in PBS with 10 mM MgCl2 and 1 mM ATP (Sigma Aldrich, A1852–1VL) for 30 min at 37 °C. CD44 was immobilized on High Capacity Neutravidin Agarose (Thermo Fisher Scientific, 29204) by incubating reactions on agarose for 1 hour at room temperature. Resin was washed on a vacuum manifold in 2 mL columns, being careful to not let the resin dry, with three successive washes of 1 mL each of RIPA, PBS with 1 M NaCl, and 50 mM ammonium bicarbonate with 2 M urea. Resins were then alkylated and digested on-bead using Preomics iST 96x kits (Preomics, P.O.00027) according to the manufacturer’s protocol and eluted peptides were dried in vacuo. Peptides were resuspended in running solvent and quantitated using a Pierce Colorimetric Peptide Quantitation Assay (Thermo Fisher Scientific, 23275). Peptides were separated and analyzed as described above, with the addition that phosphorylation (+79.97 Da) was included as a variable modification and the spectra were searched against a FASTA-formatted dataset containing the sequences of the purified proteins used.
9
Biochemical Reagents for Cell Signaling
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Rabbit anti-TRPV1 (Alomone laboratory, catalog no. ACC-030), mouse anti-Kvβ1 (Santa Cruz, catalog no. sc-373986), rabbit anti-Na+/K+ ATPase (Abcam, catalog no. ab76020), mouse anti-GST (Abclonal, catalog no. AE001), mouse anti-β-actin (Transgen Biotech, catalog no. 6609-1), mouse anti-FLAG (Sigma, catalog no. F3165), mouse anti-FLAG (MBL, catalog no. M185-3L), rabbit anti-FLAG (Proteintech, catalog no. 20543-1-AP), mouse anti-HA (MBL, catalog no. M180-3), rabbit anti-HA (Earthox, catalog no. E02218002), mouse anti-GFP (Tianjin Sungene Biotech, catalog no. KM8009), rabbit anti-GFP (WB: Solarbio, catalog no. B1025F), anti-FLAG affinity gel (Bimake, catalog no. B23100), anti-HA affinity gel (Bimake, catalog no. B23301), ProteinIso GST resin (Transgen Biotech, catalog no. DP201-01), goat anti-mouse IgG (H + L) (Jackson Immunoresearch, catalog no. 115-035-003), and goat anti-rabbit IgG (H + L) (Jackson Immunoresearch, catalog no. 111-005-003) were purchased from the indicated manufactures. The EZ-Link Sulfo-NHS-LC-Biotin (catalog no. 21335) and high-capacity NeutrAvidin-agarose (catalog no. 29202) were ordered from Thermo Fisher Scientific.
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