Biotin-4-aminophenol

Guidelines (The following is our recommended protocol. This protocol only provides guidance and should be modified according to your specific needs).
1. Cell culture^[1]
1. HeLa stable cell lines expressing APEX2-FLAG tagged proteins are cultured in DMEM medium containing 10% (v/v) fetal bovine serum and 0.6 μg/mL puromycin; HT1080 stable cell lines are cultured in MEM medium containing 10% (v/v) fetal bovine serum and 1 μg/mL puromycin.
Note: HeLa cells should be serum-starved in serum-free DMEM for 4 h before probe incubation.
2. Preparation of working solution:
1. Use 500 μM Biotin-4-aminophenol as the working concentration and apply directly in the cell incubation step.
3. Specific staining procedure:
1. Incubate the cells with 500 μM Biotin-4-aminophenol for 28 min. For subsequent 2 min EGF stimulation experiments, add 100 ng/mL EGF.
2. When 1 min remains in the probe incubation period, add 500 μM H₂O₂ to precisely trigger the proximity labeling reaction for 1 min.
3. Quench the labeling reaction using cold quenching buffer (10 mM sodium azide, 10 mM sodium ascorbate, and 5 mM Trolox dissolved in DPBS), 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 lysates and centrifuge at 4°C and 16,612×g for 10 min. Incubate 2.5 mg of cell lysate with 60 μL streptavidin agarose overnight at 4°C for subsequent experiments.
4. Afterwards, wash the streptavidin beads twice with RIPA lysis buffer, once with 2 M urea (dissolved in 10 mM Tris-HCl, pH 8.0), twice again with RIPA lysis buffer, and finally three times with 25 mM ammonium bicarbonate. Perform on-bead digestion and label-free quantitative proteomics analysis. During on-bead digestion, add dithiothreitol, iodoacetamide, and trypsin (1.5 μg). After desalting and reconstitution, the digested peptides are analyzed by nano LC-MS/MS using an Orbitrap Fusion mass spectrometer or Q Exactive HF-X equipped with Easy-nanoLC. All MS data are collected using Thermo Scientific Xcalibur software.

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