diff --git a/assays/2012_proteome_data_bmfz_stefanski/isa.assay.xlsx b/assays/2012_proteome_data_bmfz_stefanski/isa.assay.xlsx index 0107750a77bcaaa4872f982921e8c815d27eec72..9f89934b503a0623610663f6a997564712dd7680 100644 Binary files a/assays/2012_proteome_data_bmfz_stefanski/isa.assay.xlsx and b/assays/2012_proteome_data_bmfz_stefanski/isa.assay.xlsx differ diff --git a/assays/2022-04-13_proteome_discoverer/isa.assay.xlsx b/assays/2022-04-13_proteome_discoverer/isa.assay.xlsx index 7bfb15844d999d5789a44764d3419c590882cd54..b87c643593d1d09c667bff95bb7c1444cdb046fb 100644 Binary files a/assays/2022-04-13_proteome_discoverer/isa.assay.xlsx and b/assays/2022-04-13_proteome_discoverer/isa.assay.xlsx differ diff --git a/assays/2_2-FAME-analysis/isa.assay.xlsx b/assays/2_2-FAME-analysis/isa.assay.xlsx index d4152299b8cfbb3b9eec4ea4e026d93d2a0ae675..ae1b9bdb68a2d696bbdb553ec8b0d602bfd81511 100644 Binary files a/assays/2_2-FAME-analysis/isa.assay.xlsx and b/assays/2_2-FAME-analysis/isa.assay.xlsx differ diff --git a/assays/2_3-OrganellesFromEndosperm/README.md b/assays/2_3-OrganellesFromEndosperm/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/2_3-OrganellesFromEndosperm/dataset/.gitkeep b/assays/2_3-OrganellesFromEndosperm/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/2_3-OrganellesFromEndosperm/isa.assay.xlsx b/assays/2_3-OrganellesFromEndosperm/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..85f5aaa71525f08d3ad9f8bed98dd7e0ad29ea9a Binary files /dev/null and b/assays/2_3-OrganellesFromEndosperm/isa.assay.xlsx differ diff --git a/assays/2_3-OrganellesFromEndosperm/protocols/.gitkeep b/assays/2_3-OrganellesFromEndosperm/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 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 0000000000000000000000000000000000000000..a9663ad6da2ec903d10c9a9e6873d3c9b0439b49 --- /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. 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