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Surface Biotinylation Assay

Reagent​

ReagentFormulation
PBS (pH 8.0)/
SDS buffer/
Wash bufferIcecold PBS (pH 8.0) + 1 mM MgCl2 + 2.5 mM CaCl2
Sulfo-NHS-LC-BiotinWash buffer + 0.25 mg/mL Sulfo-NHS-LC-Biotin
0.1 M GlycineWash buffer + 0.1 M Glycine

Protocol​

  1. MLFs were washed three times with icecold PBS (pH 8.0) supplemented with 1 mM MgCl2 and 2.5 mM CaCl2.
  2. Sulfo-NHS-LC-Biotin (Thermo Scientific, Waltham, MA, USA) was added to the same solution at 0.25 mg mL-1 and incubated with cells at 4 °C for 30 min with gentle rocking.
  3. The unbound biotin group was quenched by the addition of 0.1 M glycine.
  4. Total proteins were extracted with lysis buffer and incubated overnight at 4 °C with NeutrAvidin agarose beads (Thermo Scientific).
  5. The beads were washed three times with PBS (pH 8.0) and bound proteins were eluted with the boiling SDS sample buffer and used for subsequent immunoblot analysis.
  6. Western Blot