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Surface Biotinylation Assay
Reagent
| Reagent | Formulation |
|---|---|
| PBS (pH 8.0) | / |
| SDS buffer | / |
| Wash buffer | Icecold PBS (pH 8.0) + 1 mM MgCl2 + 2.5 mM CaCl2 |
| Sulfo-NHS-LC-Biotin | Wash buffer + 0.25 mg/mL Sulfo-NHS-LC-Biotin |
| 0.1 M Glycine | Wash buffer + 0.1 M Glycine |
Protocol
- MLFs were washed three times with icecold PBS (pH 8.0) supplemented with 1 mM MgCl2 and 2.5 mM CaCl2.
- 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.
- The unbound biotin group was quenched by the addition of 0.1 M glycine.
- Total proteins were extracted with lysis buffer and incubated overnight at 4 °C with NeutrAvidin agarose beads (Thermo Scientific).
- 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.
- Western Blot