BS3 Crosslinker

BS3 Crosslinker is a cell-impermeable NHS ester crosslinker. BS3 Crosslinker "covalently locks" two spatially adjacent proteins on the surface of living cells into a covalent complex, and the dimerization/oligomerization status is determined by observing molecular weight shifts via Western blot. BS3 Crosslinker can crosslink EGFR and c-MET to assist in detecting and evaluating their dimerization status. It can be used in research related to oral cancer and glioblastoma^[1][2]. In Vitro:Guide (The following is our recommended protocol. This protocol serves only as a guideline and should be modified according to your specific needs.)
Assay for Detecting Receptor Dimerization on Live Cell Surfaces Using BS3 Crosslinking Method^[1][2]
1. Experimental Principle: BS3 Crosslinker is a water-soluble, membrane-impermeable homobifunctional amine-reactive crosslinker. The sulfo-NHS ester groups at its two ends undergo nucleophilic substitution reactions with the ε-amino groups of lysine residues on the surfaces of adjacent proteins, forming stable amide bonds, thereby covalently linking two protein subunits with a spatial distance ≤ 11.4 Å. The reaction is performed at 4°C, which can effectively "freeze" transient or stable receptor dimers/oligomers on the cell membrane. Subsequently, the molecular weight shift (monomer → dimer → oligomer) is detected by SDS-PAGE and immunoblotting to determine the dimerization status of the target protein.
2. Reagent Preparation:
2.1 Warm the vial: Before opening the BS3 Crosslinker vial, allow it to return to room temperature to prevent moisture entry (condensation).
2.2 Prepare fresh solution immediately: Immediately before use, dissolve BS3 Crosslinker in cold water or amine-free buffer (e.g., PBS or 20 mM HEPES, pH 7.5). Do not store stock solutions.
2.3 Buffer selection: Use buffers with a pH of 7.0-8.5 (e.g., PBS at pH 7.4). Avoid buffers containing primary amines (such as Tris or glycine) as they react with the crosslinker.
3. Reaction Procedures
3.1 Mix components: Add the freshly prepared BS3 Crosslinker solution to the protein sample to achieve a 10- to 20-fold molar excess of BS3 Crosslinker relative to the protein.
3.2 Incubation: Incubate the mixture at room temperature for 30 minutes (if the protein requires lower temperature, perform the incubation on ice).
3.3 Quench the reaction: Add a quenching buffer containing primary amines (e.g., 25-50 mM Tris, pH 7.5-8.0) to terminate the reaction, followed by incubation at room temperature for 15 minutes.
3.4 Purification/cleanup: If required for downstream experiments, remove unreacted residual BS3 Crosslinker by dialysis or gel filtration.