Biotin phenol

Manufactured by Iris Biotech

Sourced in Germany

Biotin-phenol is a chemical compound used in various laboratory applications. It functions as a labeling reagent, facilitating the detection and analysis of biomolecules through its biotin-binding properties. The core function of Biotin-phenol is to enable the labeling and subsequent identification of target molecules in research and diagnostic settings.

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Lab products found in correlation

Trolox, by Merck Group (7 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions) Sodium ascorbate, by Merck Group (4 mentions) Sodium azide, by Merck Group (3 mentions) Accunanobeads, by Bioneer (2 mentions) 2 d quant kit, by GE Healthcare (2 mentions) Sodium ascorbate, by Avantor (2 mentions) Hydrogen peroxide, by Merck Group (2 mentions) Streptavidin beads, by Thermo Fisher Scientific (2 mentions) Polyethylenimine (pei), by Polysciences (1 mentions) Click it mannaz metabolic glycoprotein labeling reagent, by Thermo Fisher Scientific (1 mentions) Rapigest, by Waters Corporation (1 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions) Proteinase k, by Roche (1 mentions) Triton x 100, by Merck Group (1 mentions) Mito v5 apex2 plasmid, by Addgene (1 mentions) Protein g beads, by Merck Group (1 mentions) Sequencing grade trypsin, by Promega (1 mentions) Streptavidin hrp, by Abcam (1 mentions)

Spelling variants of this product

Biotin-phenol reagent (3 citations) Biotin tyramide phenol (2 citations) Biotin-phenol (BP; (2 citations)

28 protocols using biotin phenol

Proximity Labeling Using dCas9-APEX2

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Proximity labeling was performed as described by Hung et al. (15 (link)). Briefly, cells were transiently transfected with polyethylenimine (Polysciences Inc., Warrington, PA, USA) according to the manufacturer’s protocols. A total of 900 ng of dCas9 plasmid DNA, 4.5 μg sgRNA plasmid DNA and 120 ng MS2-APEX2_NLS plasmid DNA were co-transfected in cells at 60–80% confluence in T75 flask. Twenty-four hours after transfection, cells were treated with 500 μM biotin-phenol (Iris Biotech GmbH, Germany) for 30 min, after which hydrogen peroxide was added to a final concentration of 1 mM and the cells were incubated for 1 min. Then, the reaction was immediately quenched by addition of quench buffer (10 mM sodium azide, 10 mM sodium ascorbate and 5 mM Trolox). Finally, the cells were either harvested directly or fixed in formaldehyde.

Qiu W., Xu Z., Zhang M., Zhang D., Fan H., Li T., Wang Q., Liu P., Zhu Z., Du D., Tan M., Wen B, & Liu Y. (2019). Determination of local chromatin interactions using a combined CRISPR and peroxidase APEX2 system. Nucleic Acids Research, 47(9), e52.

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APEX-mediated Biotin Labeling of Proteins

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Before labeling, U2OS cells were treated with the indicated reagents and biotin phenol (Iris-Biotech, 41994-02-9) containing medium was further added to 250 μM final concentration and treated for 30 min. Then 1 mM hydrogen peroxide was added to the medium to activate APEX labeling reaction for 1 min, followed by immediate quenching of reaction with ice-cold quenching buffer (1xPBS, 10 mM sodium azide, 10 mM sodium ascorbate, 2.5 mM Trolox). After four washes with cold quenching buffer, the cells were collected from plates with scrapers. Cells were lysed in lysis buffer (100 mM NaPO4, PH 8.0, 8 M Urea, 1% SDS, 10 mM sodium azide, 10 mM sodium ascorbate, 5 mM Trolox, 10 mM TCEP) and passed through an insulin syringe for 15 times to break DNA. After sonication at water bath sonicator for 10 mins, protein lysates are cleared by centrifuge. Protein concentration was measured using 2-D quant kit (GE healthcare, Cat# 80648356), by following manufacturer’ instruction. After alkylation with 20 mM iodoacetamide for 15 min, 0.5 mg of protein samples were aliquoted and equilibrated to the same volume with lysis buffer. After dilution with equal volume of ddH2O to reduce the concentration of urea to 4 M and SDS to 0.5%, the samples were incubated with streptavidin magnetic AccuNanobeads (Bioneer, Cat# TA-1015-1) at 4 °C overnight.

Lu S., Hu J., Arogundade O.A., Goginashvili A., Vazquez-Sanchez S., Diedrich J.K., Gu J., Blum J., Oung S., Ye Q., Yu H., Ravits J., Liu C., Yates JR I.I.I, & Cleveland D.W. (2022). Heat shock chaperone HSPB1 regulates cytoplasmic TDP-43 phase separation and liquid-to-gel transition. Nature cell biology, 24(9), 1378-1393.

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Biotinylation and Proteinase K Digestion

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Samples were processed essentially as described above except for biotin-phenol and proteinase K treatment. Cells were incubated with DMEM supplemented with 500 µM biotin-phenol (IrisBiotech) for 0 min, 5 min or 30 min at 4 °C or 37 °C. Subsequently, biotinylation was triggered with 1 mM H₂O₂ at room temperature or on ice for 60 s. For proteinase K digests, cells were suspended in cold homogenization buffer I (10 mM KCl, 1.5 mM MgCl2, 10 mM HEPES-KOH and 1 mM DTT, pH 7.5) and incubated shaking for 20 min at 4 °C. Cells were subsequently dounced utilizing a tight-fitting pestle on ice. Homogenates were mixed with cold homogenization buffer II (375 mM KCl, 22.5 mM MgCl2, 220 mM HEPES-KOH and 0.5 mM DTT, pH 7.5) at a ratio 1:5 (homogenization buffer I:II), centrifuged at 600 × g for 10 min and supernatants incubated with proteinase K. For mass spectrometry, samples were incubated at 37 °C for 1 h with 100 mg/ml proteinase K or 0.1% RAPIGest^TM as a control. For immunoblotting, samples were incubated at room-temperature for 30 min with 30 mg/ml proteinase K or 0.1% RAPIGest^TM as a control. Digestion was stopped by 10 mM PMSF, samples were centrifuged at 17,000 × g for 15 min and supernatants collected for lysis.

Nalbach K., Schifferer M., Bhattacharya D., Ho-Xuan H., Tseng W., Williams L.A., Stolz A., Lichtenthaler S.F., Elazar Z, & Behrends C. (2023). Spatial proteomics reveals secretory pathway disturbances caused by neuropathy-associated TECPR2. Nature Communications, 14, 870.

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Transient Transfection and RNAi Knockdown

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Transient transfections were performed using PEI (Polyethylenimine, Polysciences Europe GmbH) or Lipofectamine 2000 (Invitrogen) according to standard protocols for 12 h to 48 h. RNAi-mediated knockdown of TECPR2 was performed using Lipofectamine RNAiMAX (Invitrogen) according to manufacturer’s protocol for 48 h. The following reagents were used as indicated: ATG7 Inhibitor (Takeda, 1 µM, 24 h), Biotin-Phenol (IrisBiotech, 500 µM, 5–30 min), H₂O₂ (Sigma, 1 mM, 1 min), Proteinase K (Roche, 30 µg/ml for 30 min), RAPIGest^TM (Waters, 0.1%, 30 min), Triton X-100 (Merck, 0.2%, 30 min), PMSF (Sigma-Aldrich, 10 mM), Click-IT™ ManNAz Metabolic Glycoprotein Labeling Reagent (Thermo Scientific, 50 µM, 24 h) and Sulfo-DBCO-Biotin Conjugate (Jena Biosciences, 50 µM, 2 h). An overview of used siRNAs and plasmids can be found in Supplementary Data 9.

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APEX Induction for Proximal Proteomics

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APEX induction was carried out according to published methods [37 (link)]. Briefly, the cell medium was replaced by complete DMEM containing 500 μΜ final biotin-phenol (Iris biotech) or DMSO (control) and incubated for 30 min at 37 °C in a 5% CO₂ incubator. The APEX enzyme was induced by addition of 1 mM final H₂O₂ diluted in 1XDPBS, in the medium, for 1 min at room temperature (RT). Following the induction, the medium was aspirated and cells were washed three times with quencher solution (10 mM sodium azide, 10 mM sodium ascorbate, 5 mM Trolox in 1XDPBS). Next, cells were washed once with 1XDPBS, trypsinized, counted and used for downstream applications.

Kyriacou E, & Heun P. (2018). High-resolution mapping of centromeric protein association using APEX-chromatin fibers. Epigenetics & Chromatin, 11, 68.

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Mitochondrial Proteome Labeling by APEX2

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SK-MEL-28 cells were seeded at 70% confluency in 15-cm dishes (one per condition) and transiently transfected with 10 µg mito-V5-APEX2 plasmid (#72480; Addgene; Lam et al., 2015 (link)) using Lipofectamine 2000 (Thermo Fisher Scientific). 24 h after transfection, the medium was removed, and the cells were subsequently treated with either 1× PBS (control) or 30 µM tigecycline. 24 h after treatment, which corresponds to 48 h after transfection, cells were labeled by a 30-min incubation with 500 µM biotin-phenol at 5% CO₂, 37°C (Iris Biotech), followed by a 1-min incubation with 1 mM H₂O₂ at room temperature. Medium was quickly removed, and the biotin-phenol reaction was quenched by three washes with quencher solution (10 mM sodium ascorbate, 5 mM Trolox, and 10 mM sodium azide diluted in PBS). The cells were then also washed in 1× PBS, scraped in 4 ml cold 1× PBS, and collected for lysis. Each sample was lysed in 50 µl polysome lysis buffer supplemented with 1× Halt Protease and Phosphatase Inhibitor Single-Use Cocktail.

Vendramin R., Katopodi V., Cinque S., Konnova A., Knezevic Z., Adnane S., Verheyden Y., Karras P., Demesmaeker E., Bosisio F.M., Kucera L., Rozman J., Gladwyn-Ng I., Rizzotto L., Dassi E., Millevoi S., Bechter O., Marine J.C, & Leucci E. (2021). Activation of the integrated stress response confers vulnerability to mitoribosome-targeting antibiotics in melanoma. The Journal of Experimental Medicine, 218(9), e20210571.

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Biotin-phenol Labeling of Cells

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Cells were grown in the presence of 500 μM biotin-phenol (Iris Biotech) for 30 min at 37°C and pulsed with 1 mM H₂O₂ at RT. Biotinylation was stopped by washing three times with quencher solution (10 mM sodium azide, 10 mM sodium ascorbate, 5 mM 6-Hydroxy-2,5,7,8-tetramethylchroman2-carboxylic acid [TROLOX], DPBS). The third quenching step was performed for 15 min before washing three times with DPBS. Cells were either lysed in RIPA buffer or frozen at −80°C after adjusting cell numbers.

Siebert A., Gattringer V., Weishaupt J.H, & Behrends C. (2022). ALS-linked loss of Cyclin-F function affects HSP90. Life Science Alliance, 5(12), e202101359.

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Comprehensive Protein Biotinylation Methods

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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.

Kim D.I., Cutler J.A., Na C.H., Reckel S., Renuse S., Madugundu A.K., Tahir R., Goldschmidt H.L., Reddy K.L., Huganir R.L., Wu X., Zachara N.E., Hantschel O, & Pandey A. (2017). BioSITe: A Method for Direct Detection and Quantitation of Site-Specific Biotinylation. Journal of proteome research, 17(2), 759-769.

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Biotin-phenol Labeling Protocol

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Unless otherwise stated, reagents were obtained from Sigma-Aldrich. Phosphate buffered saline (PBS) was prepared as 18 mM sodium phosphate, 2.7 mM potassium chloride, 137 mM sodium chloride, 1.47 mM potassium phosphate, pH 7.4. Biotin-phenol (Iris Biotech), tyramide azide and tyramide alkyne were prepared as 50 mM stocks in DMSO and used in cultures at 1 mM final concentration (final DMSO concentration was 2%).

Ganapathy U.S., Bai L., Wei L., Eckartt K.A., Lett C.M., Previti M.L., Carrico I.S, & Seeliger J.C. (2018). Compartment-Specific Labeling of Bacterial Periplasmic Proteins by Peroxidase-Mediated Biotinylation. ACS infectious diseases, 4(6), 918-925.

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Proximity Labeling using AP2 Peroxidase

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The proximity labelling based on AP2 peroxidase was performed as described before^{9 (link)}. After 30 min biotin-phenol (500 µM, Iris Biotech) treatment at 37 °C, cells were supplemented with H₂O₂ (1 mM, Sigma-Aldrich) for 1 min and immediately quenched three times with cold quenching solution (10 mM sodium ascorbate, 5 mM Trolox and 1 mM sodium azide in DPBS). After 3 times more wash with PBS, the dry pellets of the harvested cells were flash frozen and stored at −80 °C.

Zhou D., Borsa M., Puleston D.J., Zellner S., Capera J., Sanderson S., Schifferer M., Hester S.S., Ge X., Fischer R., Jostins L., Behrends C., Alsaleh G, & Simon A.K. (2022). Mapping autophagosome contents identifies interleukin-7 receptor-α as a key cargo modulating CD4+ T cell proliferation. Nature Communications, 13, 5174.

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