ESO wikiadmin: Created page with "{| class="wikitable" |+ |'''Materials''' | colspan="3" | |- |Homogenisation buffer |50 mM Tris pH 7.4 |[0.5M] |▶ 10 ml |- | |2 mM MgCl₂ 8 % sucrose |[1M] |▶ 200 µl ▶8 g |- | |ddH₂O | |▶ 89.8 ml |- |TNE buffer | colspan="2" |20 mM TrisHCL pH8 [1M] |▶ 2 ml |- | | colspan="2" |130 mM NaCl [5M] |▶ 2.6 ml |- | | colspan="2" |5 mM EDTA [0.5M] |▶ 1 ml |- | | colspan="2" |10 µg/ml Aprotinin [5mg/ml] |▶ 200 µl |- | | colspan="2" |10 µg/..."

2025-02-27T12:50:05Z

Created page with "{| class="wikitable" |+ |'''Materials''' | colspan="3" | |- |Homogenisation buffer |50 mM Tris pH 7.4 |[0.5M] |▶ 10 ml |- | |2 mM MgCl2 8 % sucrose |[1M] |▶ 200 µl ▶8 g |- | |ddH2O | |▶ 89.8 ml |- |TNE buffer | colspan="2" |20 mM TrisHCL pH8 [1M] |▶ 2 ml |- | | colspan="2" |130 mM NaCl [5M] |▶ 2.6 ml |- | | colspan="2" |5 mM EDTA [0.5M] |▶ 1 ml |- | | colspan="2" |10 µg/ml Aprotinin [5mg/ml] |▶ 200 µl |- | | colspan="2" |10 µg/..."

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{| class="wikitable"
|+
|'''Materials'''
| colspan="3" |
|-
|Homogenisation buffer
|50 mM Tris pH 7.4
|[0.5M]
|▶ 10 ml
|-
|
|2 mM MgCl2

8 % sucrose
|[1M]
|▶ 200 µl

▶8 g
|-
|
|ddH2O
|
|▶ 89.8 ml
|-
|TNE buffer
| colspan="2" |20 mM TrisHCL pH8 [1M]
|▶ 2 ml
|-
|
| colspan="2" |130 mM NaCl [5M]
|▶ 2.6 ml
|-
|
| colspan="2" |5 mM EDTA [0.5M]
|▶ 1 ml
|-
|
| colspan="2" |10 µg/ml Aprotinin [5mg/ml]
|▶ 200 µl
|-
|
| colspan="2" |10 µg/ml Leupeptin [10mg/ml]
|▶ 100 µl
|-
|
| colspan="2" |1 µg/ml Pefabloc [1mg/ml]
|▶ 100 µl
|-
|
| colspan="2" |1 mM PMSF [200mM]
|▶ 500 µl
|-
|
| colspan="2" |0.5 % TritonX 100
|▶ 500 µl
|-
|
| colspan="2" |ddH2O
|▶ 92.9 ml
|-
|Sucrose
| colspan="2" |80% Sucrose: 8 g in 10 ml TNE
|
|-
|
| colspan="2" |35% Sucrose: 14 g in 40 ml TNE
|
|-
|
| colspan="2" |5% Sucrose: 1 g in 20 ml TNE
|
|}

=== Procedure ===
----

* Two days for experiment: Dissolving of sucrose in TNE at room temperature without protease inhibitors (80% needs 1 to 2 days), add inhibitors a short time before starting experiment
* Cells seeded on poly-L-lysin coated plates 1 day for experiment
* 1 h Crosslink with 1st antibody (1:1000) in 3 ml medium, 37 °C
* 3x wash with PBS
* 30 min 2nd antibody in 3 ml medium (1:500), 37°C
* 1x wash, 1 ml ice-cold homogenizing buffer on cells
* Scrape cells from plate, 15 min 7000 rpm, 4°C
* Supernatant + 4ml homogenizing buffer + 5mM EDTA (1:100) in ultracentrifuge tubes  balance out the tubes!
* 2h, 48 000 rpm, vacuum
* Discard supernatant
* Dissolving of pellet in 280 µl ice-cold TNE buffer for 10 min in cold room
* + 490 µl 80% sucrose (mixing)
* + 2500 µl 35% sucrose (careful: gradient!!!)
* + 800 µl 5% sucrose
* balance out the tubes!
* 18h 48 000 rpm, vacuum
* 8 fractions á 480 µl + 4x SDS buffer

Preparation of deterg.-resist. membrane microdomains (Sucrose Gradient) - Revision history