Measurement of endocytosis via reversible surface biotinylation: Difference between revisions
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Created page with "{| class="wikitable" |+ !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 * Incubatio..." |
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* 2 x | * 2 x 10<sup>5</sup> cells in 6 cm dishes, serum starvation over night | ||
* Wash 3x with cold PBS++ at 4°C | * Wash 3x with cold PBS<sup>++</sup> at 4°C | ||
* Incubation with Biotinylation buffer, 30 min 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 | * 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 | ||
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* Add 50 nM insulin to cells, 30 – 60 min at 37 °C, optionally add chloroquin (1 µl/ml) to avoid degradation of intracellular insulin | * 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) | * Incubation with stripping buffer (30 min at 4°C) | ||
* Wash 3x with cold PBS++ | * Wash 3x with cold PBS<sup>++</sup> | ||
* Lysis of cells (whole cell lysates) | * Lysis of cells (whole cell lysates) | ||
* Analysis via SDS Page and Western Blot | * Analysis via SDS Page and Western Blot | ||
Latest revision as of 13:53, 27 February 2025
| 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