From 71bbc099105e6f4e4f3a62c2fe64d5ffe499dcf2 Mon Sep 17 00:00:00 2001
From: Dominik Brilhaus <brilhaus@nfdi4plants.org>
Date: Mon, 8 Jan 2024 14:47:40 +0100
Subject: [PATCH] restructure

---
 .../isa.assay.xlsx                            | Bin 12013 -> 12013 bytes
 .../isa.assay.xlsx                            | Bin 6938 -> 6938 bytes
 assays/2_2-FAME-analysis/isa.assay.xlsx       | Bin 6901 -> 6901 bytes
 assays/2_3-OrganellesFromEndosperm/README.md  |   0
 .../dataset/.gitkeep                          |   0
 .../isa.assay.xlsx                            | Bin 0 -> 6908 bytes
 .../protocols/.gitkeep                        |   0
 .../protocols/2_3-OrganellesFromEndosperm.md  |   7 +++++++
 .../README.md                                 |   0
 .../dataset/.gitkeep                          |   0
 .../isa.assay.xlsx                            | Bin 0 -> 6916 bytes
 .../protocols/.gitkeep                        |   0
 .../2_5-OrganellarMembraneIsolation/README.md |   0
 .../dataset/.gitkeep                          |   0
 .../isa.assay.xlsx                            | Bin 0 -> 6911 bytes
 .../protocols/.gitkeep                        |   0
 assays/2_6-SDS-PAGE-Immunoblotting/README.md  |   0
 .../dataset/.gitkeep                          |   0
 .../isa.assay.xlsx                            | Bin 0 -> 6912 bytes
 .../protocols/.gitkeep                        |   0
 .../README.md                                 |   0
 .../dataset/.gitkeep                          |   0
 .../isa.assay.xlsx                            | Bin 0 -> 6916 bytes
 .../protocols/.gitkeep                        |   0
 .../README.md                                 |   0
 .../dataset/.gitkeep                          |   0
 .../isa.assay.xlsx                            | Bin 0 -> 6913 bytes
 .../protocols/.gitkeep                        |   0
 .../README.md                                 |   0
 .../dataset/.gitkeep                          |   0
 .../isa.assay.xlsx                            | Bin 0 -> 6923 bytes
 .../protocols/.gitkeep                        |   0
 isa.investigation.xlsx                        | Bin 8306 -> 8306 bytes
 studies/Ricinus-communis-seeds/isa.study.xlsx | Bin 7537 -> 7747 bytes
 34 files changed, 7 insertions(+)
 create mode 100644 assays/2_3-OrganellesFromEndosperm/README.md
 create mode 100644 assays/2_3-OrganellesFromEndosperm/dataset/.gitkeep
 create mode 100644 assays/2_3-OrganellesFromEndosperm/isa.assay.xlsx
 create mode 100644 assays/2_3-OrganellesFromEndosperm/protocols/.gitkeep
 create mode 100644 assays/2_3-OrganellesFromEndosperm/protocols/2_3-OrganellesFromEndosperm.md
 create mode 100644 assays/2_4-EnzymeActivityOrganellarMarkers/README.md
 create mode 100644 assays/2_4-EnzymeActivityOrganellarMarkers/dataset/.gitkeep
 create mode 100644 assays/2_4-EnzymeActivityOrganellarMarkers/isa.assay.xlsx
 create mode 100644 assays/2_4-EnzymeActivityOrganellarMarkers/protocols/.gitkeep
 create mode 100644 assays/2_5-OrganellarMembraneIsolation/README.md
 create mode 100644 assays/2_5-OrganellarMembraneIsolation/dataset/.gitkeep
 create mode 100644 assays/2_5-OrganellarMembraneIsolation/isa.assay.xlsx
 create mode 100644 assays/2_5-OrganellarMembraneIsolation/protocols/.gitkeep
 create mode 100644 assays/2_6-SDS-PAGE-Immunoblotting/README.md
 create mode 100644 assays/2_6-SDS-PAGE-Immunoblotting/dataset/.gitkeep
 create mode 100644 assays/2_6-SDS-PAGE-Immunoblotting/isa.assay.xlsx
 create mode 100644 assays/2_6-SDS-PAGE-Immunoblotting/protocols/.gitkeep
 create mode 100644 assays/2_7-ProteomeAcquisition-MassSpectrometry/README.md
 create mode 100644 assays/2_7-ProteomeAcquisition-MassSpectrometry/dataset/.gitkeep
 create mode 100644 assays/2_7-ProteomeAcquisition-MassSpectrometry/isa.assay.xlsx
 create mode 100644 assays/2_7-ProteomeAcquisition-MassSpectrometry/protocols/.gitkeep
 create mode 100644 assays/2_8-Computational-MS-DataAnalysis/README.md
 create mode 100644 assays/2_8-Computational-MS-DataAnalysis/dataset/.gitkeep
 create mode 100644 assays/2_8-Computational-MS-DataAnalysis/isa.assay.xlsx
 create mode 100644 assays/2_8-Computational-MS-DataAnalysis/protocols/.gitkeep
 create mode 100644 assays/2_9-StatisticalAnalysis-ProteomeDeconvolution/README.md
 create mode 100644 assays/2_9-StatisticalAnalysis-ProteomeDeconvolution/dataset/.gitkeep
 create mode 100644 assays/2_9-StatisticalAnalysis-ProteomeDeconvolution/isa.assay.xlsx
 create mode 100644 assays/2_9-StatisticalAnalysis-ProteomeDeconvolution/protocols/.gitkeep

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diff --git a/assays/2_3-OrganellesFromEndosperm/protocols/.gitkeep b/assays/2_3-OrganellesFromEndosperm/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/2_3-OrganellesFromEndosperm/protocols/2_3-OrganellesFromEndosperm.md b/assays/2_3-OrganellesFromEndosperm/protocols/2_3-OrganellesFromEndosperm.md
new file mode 100644
index 0000000..a9663ad
--- /dev/null
+++ b/assays/2_3-OrganellesFromEndosperm/protocols/2_3-OrganellesFromEndosperm.md
@@ -0,0 +1,7 @@
+# 2.3 Preparation of organelles from castor bean endosperm
+
+The isolation of castor bean endosperm was performed according to Cooper and Beevers (1969) and Beevers and Breidenbach (1974). The protocols were modified by using the grinding buffer as described by Reumann et al. (2007). 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 flask (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) were carefully collected, pooled, and stored at -80°C for further experiments.
+
+- Beevers, H., and Breidenbach, R. W. (1974). “Glyoxysomes,” in Methods in enzymology, Vol 16 Biomembranes, Part A. Eds. S. Fleciher and L. packer (New York London: Academic Press), 565–571.
+- Cooper, T. G., and Beevers, H. (1969). Mitochondria and glyoxysomes from castor bean endosperm. J. Biol. Chem. 244, 3507–3513. doi: 10.1016/S0021-9258(18)83401-9
+- Reumann, S., Babujee, L., Ma, C., Wienkoop, S., Siemsen, T., Antonicelli, G. E., et al. (2007). Proteome analysis of Arabidopsis leaf peroxisomes reveals novel targeting peptides, metabolic pathways, and defense mechanisms. Plant Cell 19, 3170–3193. doi: 10.1105/tpc.107.050989
\ No newline at end of file
diff --git a/assays/2_4-EnzymeActivityOrganellarMarkers/README.md b/assays/2_4-EnzymeActivityOrganellarMarkers/README.md
new file mode 100644
index 0000000..e69de29
diff --git a/assays/2_4-EnzymeActivityOrganellarMarkers/dataset/.gitkeep b/assays/2_4-EnzymeActivityOrganellarMarkers/dataset/.gitkeep
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index 0000000..e69de29
diff --git a/assays/2_4-EnzymeActivityOrganellarMarkers/isa.assay.xlsx b/assays/2_4-EnzymeActivityOrganellarMarkers/isa.assay.xlsx
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-- 
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