Biotinylation of cell surface protein was performed as described (Crupi et al., 2020 (link); Reyes-Alvarez et al., 2022 (link)). Briefly, cell surface proteins were labeled with biotin and either harvested immediately (simple cell surface biotinylation assay) or cells were treated with GDNF for the indicated times to induce RET internalization (biotinylation internalization assay). Following internalization, remaining biotin was removed from the cell surface using MeSNa stripping buffer. Cells were lysed and biotinylated proteins immunoprecipitated with streptavidin-conjugated agarose beads (Thermo Fisher Scientific) overnight at 4°C with agitation. Biotin-labeled proteins were collected by centrifugation at 2000×g, washed four times with lysing buffer, and resuspended in Laemmli buffer prior to SDS-PAGE, as previously described (Crupi et al., 2020 (link)).
Streptavidin conjugated agarose beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Streptavidin-conjugated agarose beads are a type of affinity chromatography resin. They consist of streptavidin, a protein that binds strongly to biotin, immobilized on agarose beads. These beads can be used to purify and isolate biotinylated proteins, nucleic acids, or other biomolecules from complex mixtures.
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Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Anti flag m2 mab, by Merck Group (2 mentions)
Ez link nhs ss biotin, by Thermo Fisher Scientific (2 mentions)
Glutathione (gsh), by Merck Group (2 mentions)
Biotin 16 utp, by Roche (2 mentions)
Hrp conjugated streptavidin, by Abcam (1 mentions)
Biomax cassette, by Merck Group (1 mentions)
Sulfo nhs lc biotin, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg hrp, by Beyotime (1 mentions)
Anti mouse igg hrp, by Beyotime (1 mentions)
Iodoacetamide iaa, by Merck Group (1 mentions)
Biotin peg4 alkyne, by Vector Laboratories (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Urea, by Merck Group (1 mentions)
Ammonium bicarbonate abc, by Merck Group (1 mentions)
Trypsin resuspension buffer, by Promega (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Formic acid, by Merck Group (1 mentions)
Anti mbp, by Proteintech (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
24 protocols using streptavidin conjugated agarose beads
1
Cell Surface Biotinylation and Internalization
2
Labeling Nascent Polypeptides with Biotin-Puromycin
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We labeled translating nascent polypeptides as described (15 (link)). In brief, ribosomes (4 μg of ribosomal RNA) were incubated with or without 2 μM of biotin-conjugated puromycin (Jena Bioscience) in a buffer containing 10 mM Tris (pH 7,4), 400 mM KCl and 3 mM MgCl2. After 90 min at 37°C the reaction was stopped by adding Laemmli Buffer. For treatments, ribosomes were pre-incubated with either 0.1 μg/μl RNAse A or 50 mM EDTA for 15 min at 30°C. The reaction products were analyzed by Western blot using streptavidin-conjugated HRP (Abcam). For pull-down assays, after labeling, the reaction was dialyzed against a buffer containing 10 mM Tris (pH 7,4), 300 mM KCl and 3 mM MgCl2. Then, streptavidin conjugated agarose beads (Thermo Scientific) were added, incubated for 90 min and washed with the dialysis buffer containing 0.01% NP40. The reaction products were analyzed by Western blot.
3
Affinity Capture of Biotinylated Proteins
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Samples containing proteins biotinylated with extracellular labelling were prepared by subjecting the conditioned medium to tangential flow filtration, using the 500‐kDa cutoff Biomax cassette (Millipore Sigma) with the feeding rate controlled at 4–5 ml/min, using a poly pump (Buchler) to maintain the inlet and operation pressures at <10 and <3.5 psi, respectively. The concentrated, buffer‐exchanged conditioned medium (retentate fraction) was then incubated with Streptavidin beads to eliminate unconjugated biotin‐phenol and non‐specific biotinylated proteins. The resulting precleared retentate fraction was treated with 0.2% (w/v) SDS to permeabilize membranous particles and vesicles. Both intracellular and extracellular labelling samples were then incubated for 2 h at room temperature with Streptavidin‐conjugated agarose beads (Thermo Fisher Scientific), which had been pre‐blocked with bovine serum albumin (BSA) and equilibrated with 0.2% SDS in DPBS. The beads were subsequently washed extensively with 0.2% SDS and 500‐mM sodium chloride. After the final washing with DPBS, the beads were boiled for 10 min with LDS sample buffer supplemented with 25‐mM D‐biotin to elute the biotinylated proteins. For mass spectrometry analysis, the Streptavidin beads capturing the biotinylated proteins were subjected to on‐bead trypsin digestion in the Augusta University Proteomics core.
4
Identification of RNA-binding Proteins via m1A Probes
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Biotin-labeled oligo ribonucleotides with the sequence of 5′-biotin-ACCCGUCUUGXAACACGGCCGUUGXUCACGUC-3′ (X = m1A or A) from human 28S rRNA gene and 5′-biotin-CCGUUCCGCCCXGGCCGCGCCCAGCUGGAAUGCA-3′ (X = m1A or A) from SOX18 mRNA were obtained from Integrated DNA Technologies (IDT). HEK293T or Raw264.7 cells were lysed in cell lysis and binding buffer (containing 10 mM Tris-HCl (pH 7.5), 150 mM KCl, 1.5 mM MgCl2, 0.05% (v/v) NP-40, 10 mM NaCl, 0.5 mM DTT, 0.5% (v/v) Triton X-100, 2 mM EDTA, and 0.5 units RNase inhibitor) and centrifuged at 13,000 r.p.m. at 4 °C for 15 min. The supernatant was pre-cleared at 4 °C for 1 h by incubation with streptavidin-conjugated agarose beads (Thermo Scientific). The biotinylated m1A and control baits were incubated at 4 °C with pre-cleared cell lysates for 2 h. streptavidin-conjugated agarose beads were then added to the mixture and incubated in a shaker at 4 °C for 2 h. The IP samples were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and stained with silver, different bands with intensive signal in m1A probes compared with control probes were cut and digested, and then analyzed by mass spectrometry LC-MS/MS experiments performed as previously described50 (link).
5
Cell Surface Protein Biotinylation and Isolation
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Cell surface proteins were biotinylated with 0.25 mM sulfo-NHS-LC-biotin (Thermo-Fisher Scientific, Rockford, IL) at room temperature for 30 min, followed by incubation with 100 mM glycine in PBS to quench the reaction, as previously described (Wang YN et al.22 (link)). To isolate biotinylated proteins, treated-cells were subjected to cell fractionation (see above) and the total, nuclear or cytoplasmic extracts (250 µg) were incubated overnight at 4 °C with 20 uL of Streptavidin-conjugated agarose beads (Thermo-Fisher Scientific, Rockford, IL). Beads were then collected by centrifugation at 10,000 rpm for 5 min at 4 °C, washed tree times with ice cold 0.1% Triton X-100 in PBS and the biotinylated proteins bound to the streptavidin beads were solubilized in 6X SDS-PAGE sample buffer and boiled for 10 min at 95 °C prior to western blotting.
6
Biotin-Labeling of Surface Proteins
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Adherent MEF-1 and PEA-13 cells were treated for 16 hours with or without 200 μM NOV at 37 °C. Cell surface proteins were biotinylated by incubation with 1 mg/ml EZ-Link™ Sulfo-NHS-SS-Biotin in PBS (pH 8) for 1 hour at 4 °C and quenched in 1 M Tris-Cl (pH 7.5). Cells were washed twice in PBS to remove unbound NHS-biotin and lysed in radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1% [v/v] Nonidet P-40 [NP40], 1 mM sodium deoxycholate, 1 mM PMSF, 0.05% [v/v] protease inhibitor cocktail) with gentle scraping. A second biotinylated flask (negative control) was lifted with trypsin/EDTA (to cleave surface protein interactions) followed by centrifugation at 2000 rpm for 2 minutes in a microfuge. Pelleted cells were resuspended in RIPA buffer. Biotinylated scraped or trypsinised cells were lysed for 30 minutes at 4 °C with gentle agitation. Lysates were cleared by centrifugation in a microfuge at 13000 rpm for 5 minutes at 4 °C and incubated with streptavidin conjugated agarose beads (Thermo Scientific, USA) for 1 hour at 4 °C. After centrifugation, the supernatant was discarded and purified proteins were released from the beads by boiling in 5x SDS sample buffer. Samples were resolved by SDS-PAGE and analysed by immunoblotting as described previously.
7
Click Chemistry Reagents for Bioconjugation
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DADPS biotin alkyne (catalogue no. 1331), 2‐[4‐{(bis[(1‐tert‐butyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]amino)methyl}‐1H‐1,2,3‐triazol‐1‐yl]acetic acid (BTTAA) (catalogue no. 1236), alkyne‐PEG4‐biotin (catalogue no. TA105) and alkyne‐Cy5 (catalogue no. TA116) were purchased from Click Chemistry Tools, GalNAz (catalogue no. 869186‐83‐4) from Jinan Samuel Pharmaceutical Co., Ltd, streptavidin‐conjugated agarose beads (catalogue no. 20350) from Thermo Fisher. Dithiothreitol (DTT) (catalogue no. 43815), ammonium bicarbonate (ABC) (catalogue no. 09830), iodoacetamide (IAA) (catalogue no. I1149), urea (catalogue no. U5128) and formic acid (FA; catalogue no. 06473) were purchased from Sigma Aldrich, and sequencing‐grade modified trypsin (catalogue no. V5111) and trypsin resuspension buffer (catalogue no. V542A) were purchased from Promega. All organic solvents were analytical grade or better. Antibodies included anti‐HA (CST, #2367, 1 : 3000), anti‐His (MBL, D291‐3, 1 : 10 000), anti‐MBP, (Proteintech, 15 089‐1‐AP, 1 : 10 000), anti‐streptavidin‐HRP (Beyotime, A0303, 1 : 5000), anti‐mouse IgG‐HRP (CST, #7076, 1 : 10 000) and anti‐rabbit IgG‐HRP (CST, #7074, 1 : 10 000).
8
Affinity Purification of SULT1A1 Interactors
9
Cell-Surface Protein Biotinylation and Internalization
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Biotinylation of cell-surface protein was performed as described (Crupi et al., 2020 (link); Reyes-Alvarez et al., 2022 (link)). Briefly, cell surface proteins were labelled with biotin and either harvested immediately (simple cell-surface biotinylation assay) or cells were treated with GDNF for the indicated times to induce RET internalization (biotinylation internalization assay). Following internalization, remaining biotin was removed from the cell surface using MeSNa stripping buffer. Cells were lysed and biotinylated proteins immunoprecipitated with streptavidin-conjugated agarose beads (ThermoFisher Scientific) overnight at 4°C with agitation. Biotin-labelled proteins were collected by centrifugation at 2000×g, washed 4 times with lysing buffer and resuspended in Laemmli buffer prior to SDS-PAGE, as previously described (Crupi et al., 2020 (link)).
10
Biotinylated Probe Pulldown Assay
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The biotinylated DNA (450 pmol) or RNA (500 pmol) probes and TnT proteins used for EMSA were incubated in NP-40 buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.1% NP-40, and protease inhibitor cocktail] for 3 h at 4°C with gentle rotation. DNA-protein or RNA–protein complexes were further incubated with streptavidin-conjugated agarose beads (Invitrogen) for 1 h at 4°C with gentle rotation. The pulled-down complex was washed 5 times with NP-40 buffer, then separated on an SDS-polyacrylamide gel and analyzed by immunoblot analysis.
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