ESO wikiadmin at 12:53, 27 February 2025

2025-02-27T12:53:56Z

← Older revision		Revision as of 14:53, 27 February 2025
Line 20:		Line 20:
	----		----

	* 2 x ~~105~~ cells in 6 cm dishes, serum starvation over night		* 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
Line 27:		Line 27:
	* 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

ESO wikiadmin: 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..."

2025-02-27T12:53:08Z

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..."

New page

{| 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
* 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

Measurement of endocytosis via reversible surface biotinylation - Revision history

ESO wikiadmin at 12:53, 27 February 2025