GLUT4 and CD36 on the cell surface were determined via a modified biotinylation method described previously (3 (link)). Briefly, isolated soleus muscle or L6 myotubes were stimulated with or without insulin for 50 min and then incubated in KRB buffer (±insulin) containing Sulfo-NHS-SS-Biotin (1 mg/ml) (Thermo Fisher Scientific) for 30 min. After biotinylation of cell surface proteins, the soleus muscle or L6 myotubes were rinsed with 50 mM tris-HCl buffer (pH 8.0) twice to remove free Sulfo-NHS-SS-Biotin. After lysis, tissue/cell lysates were incubated with NeutrAvidin Agarose (Thermo Fisher Scientific) at 4°C overnight. After incubation, the beads were washed three times with the ice-cold phosphate-buffered saline buffer containing 1% NP-40 to remove nonspecific binding proteins. Afterward, biotinylated cell surface proteins were eluted in SDS sample buffer at room temperature for 30 min and subjected to immunoblotting analysis. Biotinylated GLUT4 and CD36 were detected using corresponding specific antibodies.
Neutravidin agarose
Manufactured by Thermo Fisher Scientific
Sourced in United States
NeutrAvidin agarose is a modified agarose resin designed for the purification of biotin-labeled molecules. It features a high-binding capacity for biotin and can be used for affinity-based purification and separation techniques.
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Lab products found in correlation
Sulfo nhs ss biotin, by Thermo Fisher Scientific (8 mentions)
Ez link sulfo nhs ss biotin, by Thermo Fisher Scientific (5 mentions)
Ez link sulfo nhs lc biotin, by Thermo Fisher Scientific (3 mentions)
N ethylmaleimide, by Thermo Fisher Scientific (2 mentions)
Hydroxylamine nh2oh, by Thermo Fisher Scientific (2 mentions)
Sulpho nhs lc biotin, by Thermo Fisher Scientific (2 mentions)
Sulfo nhs lc biotin, by Thermo Fisher Scientific (2 mentions)
Biotin hpdp, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Cy2 conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Ez link sulfo nhs biotin, by Thermo Fisher Scientific (1 mentions)
Biotin hpdp, by Apexbio (1 mentions)
Streptavidin peroxidase, by Thermo Fisher Scientific (1 mentions)
Polyethylenimine (pei), by Merck Group (1 mentions)
Protein g sepharose, by Roche (1 mentions)
Mesna, by Thermo Fisher Scientific (1 mentions)
Complete mini edta free, by Roche (1 mentions)
Flag peptide, by Merck Group (1 mentions)
Lysis buffer, by Thermo Fisher Scientific (1 mentions)
D 1 14c mannitol, by PerkinElmer (1 mentions)
56 protocols using neutravidin agarose
1
Quantifying Cell Surface GLUT4 and CD36
2
Biotinylated Peptide Binding Assay
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Cells grown in 3.5-cm dishes were incubated with 50 μM biotinylated peptides for 30 min. Proteins were then collected in 1 ml of lysis buffer (20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 1% IGEPAL, 1 mM PMSF, protease cocktail (1:100; Cocktail III, Calbiochem), 1 mM NaF and 0.1 mM Na3VO4). Lysates were centrifuged at 11000 × g for 10 min at 4°C, and the supernatants were recovered. A 25-μl aliquot of each lysate was used to analyze the protein content, and the remaining lysate was incubated with NeutrAvidin-Agarose (Thermo Scientific, Rockford, IL, USA; Ref. 29200) for 12 h at 4°C with gentle shaking. The avidin beads bound with the peptides were collected by centrifugation (3000 × g for 1 min at 4°C). The beads were then washed five times with lysis buffer, and the bound proteins were eluted and analyzed by Western blotting. To detect biotinylated peptides, the membranes were incubated with HRP-conjugated streptavidin in TTBS (1:40000, Ref. 434323, Life Technologies) and then developed with a chemiluminescent substrate.
In parallel, the cells were incubated with 50 μM biotinylated peptides for 30 min. The cells were then washed with PBS at 4°C and fixed with 4% paraformaldehyde for 20 min. After washing, the cells were incubated with Cy2-conjugated streptavidin (1:500; Jackson ImmunoResearch, Baltimore, USA; Ref. 016-220-084) for 1 h, mounted and visualized as described previously.
In parallel, the cells were incubated with 50 μM biotinylated peptides for 30 min. The cells were then washed with PBS at 4°C and fixed with 4% paraformaldehyde for 20 min. After washing, the cells were incubated with Cy2-conjugated streptavidin (1:500; Jackson ImmunoResearch, Baltimore, USA; Ref. 016-220-084) for 1 h, mounted and visualized as described previously.
3
Biotinylation of cell surface proteins
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Cultured HUVECs were stimulated with LPS (1 μg/ml) for 4 h prior to labeling cell surface-associated proteins with EZ-Link sulfo-NHS-biotin (Thermo Scientific). Briefly, HUVEC monolayers plated on 60 mm dishes were incubated with a solution of 0.1 mg/ml of sulfo-NHS-biotin in PBS supplemented with 1.5 mM Ca2+ for 30 min at 4°C to block membrane trafficking events such as internalisation of cell surface proteins, during the biotinylation reaction. Excess sulfo-NHS-biotin was then removed by washing with cold PBS supplemented with 1.5 mM Ca2+ and cells were incubated with cold DMEM supplemented with 10% FBS to quench any remaining free reagent. Following another wash, cells were either directly lysed in situ or, for pulse-chase experiments, cultured at 37°C for the indicated times and then lysed as above. Lysates were prepared by passing cell homogenates through a 0.6 mm, 24G needle and then centrifuged at 14,000 rpm for 5 min to pellet insoluble debris. Part of the supernatant was conserved as total lysate followed by pull-down of biotinylated proteins with NeutrAvidin agarose (Thermo Scientific).
4
Proteomic Analysis of S-Nitrosylation
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Cell lysates or purified proteins were incubated with the S-methyl methanethiosulfonate (MMTS, Sigma-Aldrich) buffer (10 mM MMTS, 2.5% SDS, 100 mM Hepes, pH 7.4, 1 mM EDTA, 0.1 mM neocuproine) to block free thiol groups, and S-nitrosothiols of proteins were reduced by 20 mM ascorbate. The newly generated free thiols were biotinylated with 1 mM biotin-HPDP (APExBIO Technology). For control samples, ascorbate was omitted from the procedure. Biotinylated proteins were purified with NeutrAvidin agarose (Thermo Fisher Scientific), and the purified proteins were subjected to immunoblot analysis.
5
Biotinylated Protein Pulldown and GFP-Rac Immunoprecipitation
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The expression vectors coding for BirA-RhoB and GFP-Rac were transfected in HEK293 cells with PEI (Sigma-Aldrich) 48 h before the assays. Cells were incubated with 50 µM biotin for 24 h, lysed and subjected to pull-down assay of biotinylated proteins with Neutravidin-Agarose (Thermo Fisher Scientific) as previously described (Roux et al., 2012 (link); Rodríguez-Fraticelli et al., 2015 (link);). Alternatively, cells were lysed in 25 mM Tris-HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, and 2 mM EDTA buffer containing protease inhibitors for 1 h at 4°C. After preclearing the postnuclear supernatant for 1 h with isotype-specific control immunoglobulin (Sigma-Aldrich) conjugated to protein G–Sepharose (Sigma-Aldrich), GFP-Rac was immunoprecipitated with specific anti-GFP antibodies (Roche) conjugated to protein G–Sepharose for 12 h. In parallel, a control immunoprecipitation with the isotype-specific control immunoglobulin was performed. After extensive washes with the lysis buffer, immunoprecipitates were analyzed by Western blot with anti-Rac1 antibodies and by blot with streptavidin-peroxidase (Thermo Fisher Scientific).
6
Biotin-based surface protein analysis
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Transiently transected hRobo4-HA-FLAG dECs at 90% confluent were starved in DMEM for 7 h. Then their cell surface proteins were biotinylated with Sulfo-NHS-SS-biotin (0.25 mg/ml in DPBS, pH 7.4) at 4 °C for one hour following the suggested protocol (Thermo Fisher Scientific, #21331). The cells were further treated with Slit3 or BSA at 1 μg/ml in the presence of vehicle control DMSO or EIPA (25 μM) at room temperature for 10 min, and then for another 30 min r at 37 °C with 5% CO2. Following, the cells were lysed with RIPA buffer (no MESNA) or incubated with ice-cold 100 mM MESNA (Thermo Fisher Scientific, #50-163-8016) at 100 mM in DPBS, pH7.4 twice, 15 min each time to remove cell surface biotin. After four additional washes with ice-cold DPBS, cells were lysed with RIPA buffer (MESNA group). Biotinylated proteins were pulled down from cell lysates (both “No MESNA” and MENSA-treated samples) using Neutravidin agarose (Thermo Fisher Scientific, #29201) at 4 °C overnight and then probed with an anti-FLAG antibody in western blot. The total cell lysates were similarly analyzed.
7
Palmitoylation Detection via ABE Assay
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Palmitoylation levels of proteins were detected by using an acyl-biotinyl exchange (ABE) assay. ABE analysis of proteins was performed as published previously53 (link) with minor modifications. Dissected parietal cortex or striatum was homogenized using a polytron in lysis buffer (50 mM HEPES, 2% SDS, 1 mM EDTA) supplemented with 1% Triton, 20 mM MMTS (methyl methanethiosulfonate, Thermo Scientific) and protease inhibitors (Complete Mini EDTA-free, Roche Diagnostics). Cell debris was pelleted by centrifugation at 2000 × g and supernatant was incubated 20 min at 37 °C to block free thiols. Proteins were precipitated using acetone followed by thioester cleavage using 0.7 M of hydroxylamine (SIGMA) and labeled with 1 mM biotin-HPDP (Soltec Ventures) for 1 h at room temperature. Proteins were precipitated again with acetone. Biotinylated proteins were pulled down by binding with neutravidin-agarose (Thermo Scientific) and analyzed by western blot.
8
Quantification of Surface LH4 and GluN1
9
Histone H3.3K36me2/3 Peptide Pulldown Assay
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Peptide pull-down assay was performed as described previously with slight modifications4 (link),65 (link). Briefly, biotinylated histone peptide, which contains histone H3.3 amino acids 27–46 with di- or tri-methylated Lys-36 (i.e. H3.3K36me2/3), was incubated with NeutrAvidin agarose (Thermo Fisher, 29204) in PBS buffer for 4 h. The peptide-resin was then washed three times with 1 mL of washing buffer (PBS plus 0.1% TritonX-100) to remove unbound peptide before overnight incubation at 4 °C with the whole cell lysate prepared from 293T cells stably expressing ZM (lysate in a final IP175 buffer as described above). After binding, the resin was washed extensively in IP175 buffer for five times and analyzed by western blot.
10
Membrane Fractionation and Immunoprecipitation Protocol
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Membrane fractions were isolated as previously described [8 (link), 9 (link)]. Briefly, percol-sorbitol enriched trophozoite-infected erythrocytes were incubated at room temperature with 40 volumes of hypotonic lysis buffer (7.5mM Na2HPO4, 1mM EDTA, pH 7.5 with protease inhibitors) for 5 minutes and then subjected to a 100,000xg spin for 1 hour at 4°C to pellet membranes. Where indicated, peripheral proteins were liberated from membranes by treatment with 100mM Na2CO3 pH 11 at 4°C for 30 min, followed by a second 100,000xg spin to re-pellet membranes. Membrane pellets were then resuspended (with vigorous pipetting) in lysis buffer containing 10mM Tris-HCl pH 7.5, 1mM EDTA, 150mM NaCl with protease inhibitors and appropriate detergent at indicated concentrations (w/v). Lysates were incubated with rotation at 4°C for 1 hour, spun at 100,000xg for 1 hour or 16,000xg for 15 minutes (where indicated) to pellet insoluble material, and the resulting supernatant was used for immunoprecipitation experiments.
Immunoprecipitations were performed with 50μl of FLAG-agarose (Sigma Aldrich) or NeutrAvidin-agarose (Thermo Scientific) for 1 hour at 4°C with rotation, spun down at low speed, washed 3 times with lysis buffer, and finally eluted with FLAG peptide (Sigma Aldrich) or cleavage of the biotin spacer arm with 100mM dithiothreitol (DTT) (Sigma Aldrich).
Immunoprecipitations were performed with 50μl of FLAG-agarose (Sigma Aldrich) or NeutrAvidin-agarose (Thermo Scientific) for 1 hour at 4°C with rotation, spun down at low speed, washed 3 times with lysis buffer, and finally eluted with FLAG peptide (Sigma Aldrich) or cleavage of the biotin spacer arm with 100mM dithiothreitol (DTT) (Sigma Aldrich).
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