diff --git a/assays/EnzymaticCarbohydrateDigestionAndTLC/isa.assay.xlsx b/assays/EnzymaticCarbohydrateDigestionAndTLC/isa.assay.xlsx index 3f06afef11bc5fd0acf1227f5a332ce28cd74944..fdc14a22e6e13bdd67e5725adec461718cf41fe2 100644 Binary files a/assays/EnzymaticCarbohydrateDigestionAndTLC/isa.assay.xlsx and b/assays/EnzymaticCarbohydrateDigestionAndTLC/isa.assay.xlsx differ diff --git a/assays/HeterologousProteinProductionAndPurification/README.md b/assays/HeterologousProteinProductionAndPurification/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/HeterologousProteinProductionAndPurification/dataset/.gitkeep b/assays/HeterologousProteinProductionAndPurification/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/HeterologousProteinProductionAndPurification/isa.assay.xlsx b/assays/HeterologousProteinProductionAndPurification/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..d84e333c8baff6f9e4db2e7c2cf8fb19085b70ac Binary files /dev/null and b/assays/HeterologousProteinProductionAndPurification/isa.assay.xlsx differ diff --git a/assays/HeterologousProteinProductionAndPurification/protocols/.gitkeep b/assays/HeterologousProteinProductionAndPurification/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/HeterologousProteinProductionAndPurification/protocols/HeterologousProteinProductionAndPurification.md b/assays/HeterologousProteinProductionAndPurification/protocols/HeterologousProteinProductionAndPurification.md new file mode 100644 index 0000000000000000000000000000000000000000..7efd982a524270fdb814badf91e24ca50a1fdd30 --- /dev/null +++ b/assays/HeterologousProteinProductionAndPurification/protocols/HeterologousProteinProductionAndPurification.md @@ -0,0 +1,2 @@ +## Heterologous protein production and purification from N. benthamiana +*A. tumefaciens* GV3101::pMP90RK strains carrying the binary vectors for protein production (antibiotic selection: 30 μg·mL-1 Rifampicin, 25 μg·mL-1 Kanamycin, 50 μg·mL-1 Carbenicillin) and *A. tumefaciens* GV3101 strains carrying the binary vector for viral p19 silencing inhibitor expression (antibiotic selection: 30 μg·mL-1 Rifampicin, 30 μg·mL-1 Gentamicin, 100 μg·mL-1 Carbenicillin) were grown in selection LB liquid medium at 28 °C, 180 rpm for three days. The cultures were centrifuged (3,500 g for 15 min), resuspended in infiltration buffer (10 mM MES pH 5.5, 10 mM MgCl2, 200 μM acetosyringone) to an OD600 of 1 and incubated for 1 h in the dark at 28 °C, 180 rpm. Each of the two *A. tumefaciens* strains carrying the GBP1 production constructs was mixed with the *A. tumefaciens* strain carrying the p19-expressing construct in a 1:1 ratio. The bacterial suspensions were infiltrated into the four youngest, fully developed leaves of four-week-old *N. benthamiana* plants with a needleless syringe. Five days after infiltration, the leaves were detached from the plant and ground in liquid nitrogen. Protein purification was carried out according to Werner and coworkers84 with minor modifications: The ground plant material (up to the 5 mL mark of 15-mL tube) was thoroughly resuspended in 5 mL of ice-cold extraction buffer (100 mM Tris pH 8.0, 100 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 10 mM DTT, 100 μg·mL-1 Avidin) and centrifuged at 10,000 g, 4 °C for 10 min. The supernatant was filtered through a PD-10 desalting column (Sigma-Aldrich, Taufkirchen, Germany), transferred to a new tube, and supplemented with 75 μL·mL-1 Strep-Tactin Macroprep (50% slurry) (IBA Lifesciences GmbH, Göttingen, Germany). Samples were incubated in a rotary wheel at 4 °C for 1 h, followed by centrifugation for 30 s at 700 g. The supernatant was discarded, and the beads were washed three times with 2 mL of washing buffer (50 mM Tris pH 8.0, 100 mM NaCl, 0.5 mM EDTA, 0.005% Triton X-100, 2 mM DTT). Proteins were eluted from the beads by adding 100 μL of elution buffer (wash buffer containing 10 mM biotin) and incubating at 800 rpm for 5 min at 25 °C. The samples were centrifuged at 700 g for 20 s and the elution was repeated two more times. The elution fractions were pooled and dialyzed overnight against cold Milli-Q water (dialysis tubing with 6-8 kDa cut-off). Proteins were stored on ice at 4 °C for further use. The success of protein purification was analyzed by SDS PAGE and Western Blotting. \ No newline at end of file diff --git a/assays/MALDI-TOFAnalysis/isa.assay.xlsx b/assays/MALDI-TOFAnalysis/isa.assay.xlsx index 20dcdadb1fa483b53a36bf9f666de13b83249f53..0e7fda2bd3a89fb0d5f52990f71bd8f5ed93da8b 100644 Binary files a/assays/MALDI-TOFAnalysis/isa.assay.xlsx and b/assays/MALDI-TOFAnalysis/isa.assay.xlsx differ diff --git a/assays/MS-MSAnalysisOfThePull-downProteins/isa.assay.xlsx b/assays/MS-MSAnalysisOfThePull-downProteins/isa.assay.xlsx index e255f776b0d2aef61137b91430abac9d5c467b29..4546654fcf73c8ae698ff6f7f205eed161ee7728 100644 Binary files a/assays/MS-MSAnalysisOfThePull-downProteins/isa.assay.xlsx and b/assays/MS-MSAnalysisOfThePull-downProteins/isa.assay.xlsx differ diff --git a/assays/OxidativeBurstAssay/isa.assay.xlsx b/assays/OxidativeBurstAssay/isa.assay.xlsx index 3a39c6cc0105d7e7e397bed27978e10513ea729b..8a5169920669e5618f932387409e080b73d90f31 100644 Binary files a/assays/OxidativeBurstAssay/isa.assay.xlsx and b/assays/OxidativeBurstAssay/isa.assay.xlsx differ diff --git a/assays/QuantificationAndStatisticalAnalyses/isa.assay.xlsx b/assays/QuantificationAndStatisticalAnalyses/isa.assay.xlsx index 21d2b47748d1d686afc50d27a3b7293ff4bfe7ec..606610f59b59d290c9a13fea639b7f01b46105df 100644 Binary files a/assays/QuantificationAndStatisticalAnalyses/isa.assay.xlsx and b/assays/QuantificationAndStatisticalAnalyses/isa.assay.xlsx differ diff --git a/assays/qRT-PCR/isa.assay.xlsx b/assays/qRT-PCR/isa.assay.xlsx index afce164c9ec1c88bc5a26d8ae15e3b103860f45e..8396522a05726347aeaba9edfc3a1aa3f8bef637 100644 Binary files a/assays/qRT-PCR/isa.assay.xlsx and b/assays/qRT-PCR/isa.assay.xlsx differ diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx index 679358f2e2a6d22071925611204bdec1cff8f714..35d92118e1d1d63b91363a457cb3c6b985dde5ce 100644 Binary files a/isa.investigation.xlsx and b/isa.investigation.xlsx differ diff --git a/studies/Barley/isa.study.xlsx b/studies/Barley/isa.study.xlsx index e66c2ddd120d452162c8cc2f05df705a9f1549aa..3b88dfa48a1092ebc4f975a42dd6918f5d37a3d3 100644 Binary files a/studies/Barley/isa.study.xlsx and b/studies/Barley/isa.study.xlsx differ diff --git a/studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/isa.study.xlsx b/studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/isa.study.xlsx index bcfdbbabd0555ff617009264ec108a524e80e3c2..7bf8c2deb5128c609e331e4398e810aacb3dc1de 100644 Binary files a/studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/isa.study.xlsx and b/studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/isa.study.xlsx differ diff --git a/studies/NicotianaBenthamiana/isa.study.xlsx b/studies/NicotianaBenthamiana/isa.study.xlsx index 989f05098c9f5b33ef1d701539b5c5fe39911ba3..191640abad08d48c42764ba3637a46350566a9d8 100644 Binary files a/studies/NicotianaBenthamiana/isa.study.xlsx and b/studies/NicotianaBenthamiana/isa.study.xlsx differ diff --git a/studies/PlantMaterialAndGrowthConditions/isa.study.xlsx b/studies/PlantMaterialAndGrowthConditions/isa.study.xlsx index 438c42b241eb16534b10c2217e9d0950499862e1..7b3a4866c50feea27cf08bae22b6925e28709f08 100644 Binary files a/studies/PlantMaterialAndGrowthConditions/isa.study.xlsx and b/studies/PlantMaterialAndGrowthConditions/isa.study.xlsx differ