Measurement of endocytosis via reversible surface biotinylation
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| Materials | |
|---|---|
| Biotinylation buffer: | PBS++ (pH 7.4) + 400 µg/ml Sulfo-NHS-SS- Biotin (freshly added) |
| Quenching buffer: | PBS++ (pH 7.4) + 100 mM Glycin |
| Stripping buffer: | 50 mM Tris (pH 8.6), 100 mM NaCl, freshly added: 0.2 % BSA, 100 mM MESNa |
| Serum starvation medium: | DMEM + 0.5 % calf serum (CS) |
Procedure
- 2 x 105 cells in 6 cm dishes, serum starvation over night
- Wash 3x with cold PBS++ at 4°C
- Incubation with Biotinylation buffer, 30 min at 4 °C
- Excessive biotin has to react with Quenching buffer (1x 20 min at 4 °C), 1x 5 min during transport from cold room to cell culture
- Add 10 ml serum starvation medium to cells
- Add 50 nM insulin to cells, 30 – 60 min at 37 °C, optionally add chloroquin (1 µl/ml) to avoid degradation of intracellular insulin
- Incubation with stripping buffer (30 min at 4°C)
- Wash 3x with cold PBS++
- Lysis of cells (whole cell lysates)
- Analysis via SDS Page and Western Blot