Biotin-4-aminophenol 

Biotin-4-aminophenol is a biotin-phenol analog. Biotin-4-aminophenol generates free radicals and conjugates to tyrosine residues in proteins more efficiently and selectively than the previously reported BP1^[1].

1. Cell culture^[1]
1) HeLa stable cell line expressing APEX2-FLAG tagged protein was cultured in DMEM medium containing 10% (v/v) fetal bovine serum and 0.6 μg/mL puromycin; HT1080 stable cell line was cultured in MEM medium containing 10% (v/v) fetal bovine serum and 1 μg/mL puromycin.
Note: HeLa cells need to be starved in serum-free DMEM for 4 h before incubation with probes.
2. Preparation of working solution:
1) Use 500 μM Biotin-4-aminophenol as the working solution concentration and use it directly in the cell incubation step.
3. Specific staining steps:
1) Incubate cells with 500 μM Biotin-4-aminophenol for 28 min, and then add 100 ng/mL EGF if a 2 min EGF stimulation experiment is performed.
2) When there is 1 min left in the probe incubation time, add 500 μM H₂O₂ to precisely trigger the proximity labeling reaction for 1 min.
3) Quench the labeling reaction with cold quenching buffer (10 mM sodium azide, 10 mM sodium ascorbate, 5 mM tocotrienols, dissolved in DPBS), and then wash the cells three times with ice-cold DPBS. Lyse the cells with 700 μL ice-cold RIPA lysis buffer. After lysis, sonicate the cell lysate and centrifuge at 4°C, 16,612×g for 10 min. 2.5 mg of cell lysate was incubated with 60 μL of streptavidin agarose overnight at 4°C for subsequent experiments.
4) Afterwards, the streptavidin beads were washed twice with RIPA lysis buffer, once with 2 M urea (dissolved in 10 mM Tris-HCl, pH 8.0), twice with RIPA lysis buffer, and finally three times with 25 mM ammonium bicarbonate. On-bead digestion and label-free quantitative proteomics analysis were performed. For on-bead digestion, dithiothreitol, iodoacetamide, and trypsin (1.5 μg) were added. The digested peptides were desalted, resolubilized, and analyzed by nano LC-MS/MS on an Orbitrap Fusion mass spectrometer equipped with Easy-nanoLC or Q Exactive HF-X. All MS data were collected using Thermo Scientific Xcalibur software.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

335.42

C₁₆H₂₁N₃O₃S

901770-40-9

Solid

White to off-white

OC(C=C1)=CC=C1NC(CCCC[C@H]2[C@]3([H])[C@](NC(N3)=O)([H])CS2)=O

Room temperature in continental US; may vary elsewhere.

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month. When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Ke M, et al. Spatiotemporal profiling of cytosolic signaling complexes in living cells by selective proximity proteomics. Nat Commun. 2021;12(1):71.  [Content Brief]