The cell surface biotinylation assay was performed as previously described21 , 22 with some modifications. Briefly, the VSMCs were cultured to 90% confluence in 60‐mm dishes. After washing them with ice‐cold PBS, cell surface proteins were biotinylated by incubation with 2 mg/mL of EZ‐Link sulfo‐NHS‐SS‐biotin (21331; Thermo Fisher) in PBS for 2 hours at 4°C. The cells were incubated with a medium containing AT1‐AA‐positive IgG (1 μmol/L) or Ang II (1 μmol/L) at 37°C for 30 minutes to receptor internalization. The remaining biotin on the cell surface was cleaved by incubation with cutting buffer (20 mmol/L dithiothreitol and 15 mmol/L glycine in PBS) for 2 hours at 4°C. After washing them 3 times with ice‐cold PBS, cells were harvested in a 500 μL radioimmunoprecipitation assay lysis buffer, 50 μL lysis samples were separated for input, and the rest were incubated with high‐capacity streptavidin agarose (20359; Thermo Fisher) for 1 hour at 4°C to precipitate biotinylated proteins. Finally, the collected proteins were washed with ice‐cold PBS and they were eluted from the beads by boiling in 2× loading buffer. Then, they were separated by SDS‐PAGE, and they were immunoblotted with an AT1R antibody.
High capacity streptavidin agarose
Manufactured by Thermo Fisher Scientific
Sourced in United States
High-capacity streptavidin agarose is a solid-phase matrix designed for the capture and purification of biotinylated molecules. Streptavidin, a bacterial protein with a high affinity for biotin, is covalently immobilized on agarose beads, providing a robust platform for affinity-based separations.
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Lab products found in correlation
Ez link sulfo nhs ss biotin, by Thermo Fisher Scientific (2 mentions)
Ms grade trypsin from porcine pancreas, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Iodoacetamide, by Merck Group (2 mentions)
Trifluoroacetic acid, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Biotin, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Biotin azide, by Thermo Fisher Scientific (1 mentions)
Anti il 17a antibody, by R&D Systems (1 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Rhadam17, by R&D Systems (1 mentions)
Synergy h4, by Agilent Technologies (1 mentions)
Lc ms grade acetonitrile, by Merck Group (1 mentions)
Fp biotin, by Thermo Fisher Scientific (1 mentions)
Urea, by Merck Group (1 mentions)
Streptavidin agarose, by Thermo Fisher Scientific (1 mentions)
Dynabeads m 280, by Thermo Fisher Scientific (1 mentions)
15 protocols using high capacity streptavidin agarose
1
Cell Surface Biotinylation Assay
2
IL-17A Covalent Labeling and Detection
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0.5 μM full length IL-17A (R&D Systems) was pre-incubated with DMSO or 50 μM a control compound in PBS at room temperature for 30 min, after which 5 μM sulfonyl fluoride probe compound 4 was added to the protein mixture (final sample volume 50 μl) and incubated for 1 h. Click chemistry was then performed at room temperature for 2 h. Briefly, 38.2 μL of 4% SDS in HEPES buffer (pH 7.5) was added to the sample, followed by the addition of 1.9 μL of 4 mM biotin azide (Life Technologies), 2 μL of 50 mM CuSO4, 5.9 μL TBTA in DMSO:tert-BuOH (1:5), and 2 μL of 50 mM TCEP (final concentrations: 1.5% SDS, 75 μM biotin azide, 1 mM CuSO4, 5% tert-BuOH and 1 mM TCEP). 1 mL of 6 M urea in PBS was added to quench the reaction. The mixture was then incubated with 100 μL high-capacity streptavidin agarose (Thermo Fisher Scientific) at room temperature for 2 h with rotation. Beads were centrifuged at 1000 g for 1 minute and washed 3 times with 1 mL 4 M urea in PBS. Proteins were eluted by adding 90 μL of 2× LDS sample buffer (Life Technologies) and heated at 95 °C for 10 minutes. Eluates were separated on a 12% NuPAGE Bis-Tris gel, transferred to a PVDF membrane, and then analyzed by immunoblot (anti-IL17A antibody from R&D Systems).
3
Proteomic Analysis of Histone H4 Modifications
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High-capacity streptavidin agarose (Thermo Scientific) was incubated with C-terminal biotin-tagged 20 amino acid N-terminal peptides of H4, H4R3me2a, and H4R3me2s peptide immunoprecipitates from nuclear extracts of HCT116 cells, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and stained with SimplyBlue Safestain (Invitrogen). Protein bands of interest were excised and subjected to electrospray–ion trap tandem mass spectrometry (LCQ-Deca, Finnigan).
4
Quantifying ADAM17 Catalytic Activity
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Catalytic activity of the rhADAM17 (100 ng/ml, purchased from R&D Systems, MN, USA) was measured by hydrolytic processing of Fluorogenic Peptide Substrate III Mca-P-L-A-Q-A-V-Dpa-R-S-S-S-R-NH2 (20 µM, R&D Systems, MN, USA) followed by monitoring the increasing fluorescence intensity at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 10, 30 and 120 min at 37 °C with a Synergy H4 plate reader (BioTek, VT, USA). All reactions were performed in a fluorogenic buffer containing 25 mM Tris, pH 9.5, 150 mM NaCl, 2.5 µM ZnCL2 and 0.005% Brij-35. For the rhADAM17 in vitro CA IX cleavage activity evaluation, the FL-CA IX-C-SBP protein was immobilised to High-Capacity Streptavidin Agarose (Thermo Fisher Scientific, MA, USA), washed and incubated with recombinant ADAM17 (dissolved at a concentration of 100 ng/ml in 25 mM Tris, pH 9.5, containing 2.5 µM ZnCL2 and 0.005% Brij) for 3 h at 37 °C. Thereafter, the supernatant was analysed by ELISA as described below.
5
Serine Hydrolase Profiling Protocol
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ABPP ActivX Serine Hydrolase Probes ActivX TAMRA FP (cat no. 88318) and FP-Biotin (cat no. 88317), Zeba spin desalting columns, 5 ml (cat no. 89892), high-capacity streptavidin agarose (cat no. 20349) were purchased from Thermo Fisher Scientific (United States). Dimethyl sulfoxide (Cat no. D2650), Dithiothreitol (cat no. D9779), Iodoacetamide (cat no. I6125), MS grade trypsin from porcine pancreas (cat no. T6567), Trifluoroacetic acid (cat no. T6508) were purchased from Sigma Aldrich (United States). LCMS grade acetonitrile was procured from Sigma Aldrich. Urea was ordered from Merck.
6
Streptavidin-Based Protein Pulldown Assays
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We used two streptavidin-agarose resins: high-capacity streptavidin-agarose (20359, ThermoFisher Scientific, Waltham, MA, USA) and streptavidin agarose (15942-050, Invitrogen, Waltham, MA, USA), which is the same product as currently available SA100-04. The resins were mixed well before being taken (20 μL × sample numbers) into a microcentrifuge tube, washed once with 1–1.5 mL of washing buffer (25 mM Tris, pH7.4, 15 mM NaCl, 1% NP40, and 0.5% sodium deoxycholate), and resuspended in (20 μL × sample numbers) of binding buffer A (20 mM Tris pH7.4, 300 mM KCl, 0.2 mM EDTA. 1.5 mM MgCl2, and 0.5 mM PMSF). Streptavidin magnetic beads (S1420S New England Biolabs (ΝΕΒ, Ipswich, MA, USA) and Dynabeads M-280 Invitrogen, Waltham, MA, USA) were also tested for IRE-IRP pull-down assays. These beads were magnetized with a DynaMag-2 magnetic stand during washing with NEB magnetic beads buffer (20 mM Tris-HCl pH7.5, 0.5 M NaCl, and 1 mM EDTA) or Dynabeads buffer (5 mM Tris-HCl pH7.5, 1 M NaCl, and 0.5 mM EDTA).
7
Identification of hARTC1 and hARTC3 Interactors
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To find hARTC1- and hARTC3-binding partners, we harvested HEK-293T cells stably expressing SFB-hARTC1 and SFB-hARTC3 and washed cells with phosphate-buffered saline (PBS). Cells were lysed with 30 ml of ice-cold NETN 100 buffer (100 mM NaCl, 20 mM Tris–HCl, pH 8.0, 0.5 mM EDTA-2Na, 0.5% NP-40). The pellets were incubated with NETN 300 buffer (300 mM NaCl, 20 mM Tris–HCl, pH 8.0, 0.5 mM EDTA-2Na, 0.5% NP-40). The soluble fraction was incubated with 0.5 ml High Capacity Streptavidin Agarose (Thermo, 20359) beads for 4 h. The beads were washed three times with NETN 100 buffer. Associated proteins were eluted with 2 mM Biotin (Sigma, B4501) in PBS and further incubated with 0.05 ml S beads (MERCK, 69704). The bound proteins were eluted with sodium dodecyl sulfate (SDS) loading buffer and subjected to 10% SDS–polyacrylamide gel electrophoresis (PAGE). The entire protein band (<1 cm) was excised and analyzed by MS.
8
Serine Hydrolase Profiling using ActivX Probes
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ABPP ActivX Serine Hydrolase Probes ActivX TAMRA-FP (cat no. 88318) and ActivX Desthiobiotin-FP (cat no. 88317), Zeba spin desalting columns, 5 ml (cat no. 89892), high capacity streptavidin agarose (cat no. 20349), anti-TAMRA monoclonal antibody (cat no. MA1-041), cDNA Synthesis Kit, restriction endonucleases and T4 DNA ligase enzyme were procured from Thermo Scientific (USA). Dimethyl Sulfoxide (Cat no. D2650), Dithiothreitol (cat no. D9779), Iodoacetamide (cat no. I6125), MS grade trypsin from porcine pancreas (cat no. T6567), Trifluoroacetic acid (cat no. T6508) were purchased from Sigma Aldrich (USA). LCMS grade acetonitrile (cat no. 9829-03) and water were procured from JT baker Avantor. Urea (cat no.194857) was ordered from MP Biomedicals, Mumbai, India.
9
Biotinylation and Enrichment of Cell Surface Proteins
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Transfected cells were cooled down to 4°C and washed with PBS-CM (0.1 mM CaCl2 and 1 mM MgCl2 in PBS). Biotin solution (1 mg/ml EZ-Link Sulfo-NHS-SS-Biotin (ThermoFisher) in PBS-CM) was added for 30 min at 4°C. Afterwards, cells were incubated with quenching buffer (50 mM TRIS, pH 8 in PBS-CM) for 10 min at 4°C and washed three times with PBS-CM. Cells were then harvested in PBS-CM and lysed (50 mM TRIS, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, cOmplete protease inhibitor (Roche), pH 7.4). The total protein amount was determined using a BCA protein assay kit (ThermoFisher) and sufficient amount of lysate was taken for control Western blot analysis. The remaining lysate was incubated with washed High Capacity Streptavidin Agarose (ThermoFisher) for 1 h at 4°C to pull down biotinylated cell surface proteins. The agarose beads were washed three times with lysis buffer. Then, 80 μl 1X reducing sample buffer was added and samples were heated for 30 min at 60°C to separate precipitated proteins from agarose beads. Whole lysates and precipitated proteins were then analyzed by Western blot.
10
Histone H3 Peptide Interactome
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C‐terminal biotin‐tagged 19 amino acid N‐terminal peptides of H3, H3K4me1, and H3K4me3 were incubated with nuclear extract of SW1990 cells, and then with high‐capacity streptavidin agarose (Thermo Scientific, USA) for immunoprecipitation. The product was separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) with silver staining. Protein bands of interest were excised and subjected to electrospray‐ion trap tandem mass spectrometry (LCQ‐Deca, Finnigan).
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