From 8bb715c47451ef1b8b518bc1a72a578d94370a0b Mon Sep 17 00:00:00 2001
From: Dominik Brilhaus <dominik.brilhaus@hhu.de>
Date: Tue, 12 Apr 2022 11:43:52 +0200
Subject: [PATCH] update isa.assay

---
 _DominikNotes/2022-04-12_isa.assay.xlsx       | Bin 0 -> 18247 bytes
 .../isa.assay.xlsx                            | Bin 16188 -> 18247 bytes
 .../protocols/02_organelle_prep.md            |   8 ++++++++
 3 files changed, 8 insertions(+)
 create mode 100644 _DominikNotes/2022-04-12_isa.assay.xlsx

diff --git a/_DominikNotes/2022-04-12_isa.assay.xlsx b/_DominikNotes/2022-04-12_isa.assay.xlsx
new file mode 100644
index 0000000000000000000000000000000000000000..ff8e34df1423bc317e2f3be19ac62f7219c20603
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diff --git a/assays/2012_proteome_data_bmfz_stefanski/isa.assay.xlsx b/assays/2012_proteome_data_bmfz_stefanski/isa.assay.xlsx
index beb998a4158e4d3477a46c5fdf117638b0dff200..ff8e34df1423bc317e2f3be19ac62f7219c20603 100644
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diff --git a/assays/2012_proteome_data_bmfz_stefanski/protocols/02_organelle_prep.md b/assays/2012_proteome_data_bmfz_stefanski/protocols/02_organelle_prep.md
index e9f37d3..c82cfb6 100644
--- a/assays/2012_proteome_data_bmfz_stefanski/protocols/02_organelle_prep.md
+++ b/assays/2012_proteome_data_bmfz_stefanski/protocols/02_organelle_prep.md
@@ -2,3 +2,11 @@
 # Preparation of organelles from castor bean endosperm
 
 The isolation of castor bean endosperm was performed according to Cooper & Beevers (1969) and Beevers & Breidenbach (1974) {Cooper:1969tz} {Beevers:1974ug}. The protocols were modified by using the grinding buffer as described by Reumann et al. (2007) {Reumann:2007wi}. All steps were carried out on ice in a cold room (4°C) unless indicated otherwise. 30 g of endosperm tissue from 5-day old dark-grown Ricinus seedlings was harvested by removing the yellow cotyledons using the blunt side of a scalpel blade. The resulting endosperm was chopped using an onion chopper in 60 mL grinding buffer (170 mM Tricine pH X (KOH), 1 M Sucrose, 1% (w/v) BSA, 10 mM KCl, 1 mM MgCl2, 2 mM EDTA, 0,5 % (w/v) PVP-40, and 5 mM DTT). The suspension was further homogenized using mortar and pestle. The homogenate was filtered through four layers cheesecloth. The crude extract was centrifuged at 1,200 g for 10 minutes to remove cell debris. The supernatant was carefully decanted into a new flasks (approx. 40 mL). To separate the organelles, 6 mL of the obtained extract was loaded onto the top of a discontinuous sucrose gradient prepared in 20 mM Tricine-KOH (pH 7.5) and 1 mM EDTA. The density gradient consists of the following sucrose steps (from top to bottom): 5 ml 30% (w/w) sucrose, 3 ml 44% (w/w) sucrose, 5 ml 48% (w/w) sucrose, 5 ml 49% (w/w) sucrose, 1 ml 50% (w/w) sucrose, 2 ml 54% (w/w) sucrose, and 2 ml 60% (w/w) sucrose. The organelles were separated by ultracentrifugation at 105,026 g using a swing-out rotor for 3 hours. Four visible bands at the interface 30% − 44% (w/w) sucrose solution (fraction 1), 44% − 48% (w/w) sucrose solution (fraction 2), 48% − 49% (w/w) sucrose solution (fraction 3), and 50% − 54% (w/w) sucrose solution (fraction 4) was carefully collected, pooled and stored at -80°C for further experiments.
+
+
+
+Glyoxysomes: F4
+proplastids: F3
+mitochondria: F2
+Endoplasmic Reticulum (ER) and Golgi: F1
+
-- 
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