From 59fa9a0e14ee4630b8ef3ad02409bd0d793721a1 Mon Sep 17 00:00:00 2001 From: Viktoria Petrova <vipet103@hhu.de> Date: Mon, 8 Jan 2024 19:27:07 +0100 Subject: [PATCH] add references to protocols --- .../HeterologousProteinProductionAndPurification.md | 2 +- .../protocols/PreparationOfThePlantMaterial.md | 5 +++++ assays/OxidativeBurstAssay/protocols/ROSBurstAssay.md | 2 +- .../protocols/ProteinPull-downWithBiotinylatedLaminarin.md | 2 ++ 4 files changed, 9 insertions(+), 2 deletions(-) create mode 100644 assays/OxidativeBurstAssay/protocols/PreparationOfThePlantMaterial.md diff --git a/assays/HeterologousProteinProductionAndPurification/protocols/HeterologousProteinProductionAndPurification.md b/assays/HeterologousProteinProductionAndPurification/protocols/HeterologousProteinProductionAndPurification.md index 7efd982..be16729 100644 --- a/assays/HeterologousProteinProductionAndPurification/protocols/HeterologousProteinProductionAndPurification.md +++ b/assays/HeterologousProteinProductionAndPurification/protocols/HeterologousProteinProductionAndPurification.md @@ -1,2 +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 +*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 coworkers (Werner et al., 2008) 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/OxidativeBurstAssay/protocols/PreparationOfThePlantMaterial.md b/assays/OxidativeBurstAssay/protocols/PreparationOfThePlantMaterial.md new file mode 100644 index 0000000..28d2f56 --- /dev/null +++ b/assays/OxidativeBurstAssay/protocols/PreparationOfThePlantMaterial.md @@ -0,0 +1,5 @@ +## Preparation of plant material - **Barley** +For immunity assays in barley, the roots and shoots of seven-day-old seedlings were separated. The root tissue between 2 cm below the seed and 1 cm above the tip was cut into 5 mm pieces. Each assay was carried out with randomly selected root pieces from 16 barley seedlings. Four root pieces were transferred to each well of a 96-well microtiter plate containing 150 μL of sterile Milli-Q water. Barley shoot assays were performed on 3-mm leaf discs punched from the youngest leaves of eight individual barley seedlings. + +## Preparation of plant material - *N. benthamiana* +For immunity assays in N. benthamiana, 3-mm leaf discs from the youngest, fully developed leaf of eight three-week-old plants were transferred to a 96-well plate filled with 150 μL of sterile Milli-Q water. \ No newline at end of file diff --git a/assays/OxidativeBurstAssay/protocols/ROSBurstAssay.md b/assays/OxidativeBurstAssay/protocols/ROSBurstAssay.md index 57a3a5a..75b8c21 100644 --- a/assays/OxidativeBurstAssay/protocols/ROSBurstAssay.md +++ b/assays/OxidativeBurstAssay/protocols/ROSBurstAssay.md @@ -1,2 +1,2 @@ ## ROS burst assay -The ROS burst assay was based on previously published protocols.33,85 In brief, a 96-well plate containing water and plant material (as described above) was incubated overnight at RT to remove ROS that had resulted from mechanical damage to the tissue during preparation. The next day, the water was replaced with 100 μL of fresh Milli-Q water containing 20 μg·mL-1 horseradish peroxidase (Sigma-Aldrich, Taufkirchen, Germany) and 20 μM L-012 (Wako Chemicals, Neuss, Germany). After a short incubation period (∼15 min), 100 μL of double-concentrated elicitor solutions were added to the wells. All elicitors were dissolved in Milli-Q water without additional treatment. Measurements of elicitor-triggered apoplastic ROS production were started immediately and performed continuously with an integration time of 450 ms in a TECAN SPARK 10 M multiwell plate reader (Männedorf, Switzerland). +The ROS burst assay was based on previously published protocols (Chandrasekar et al., 2022; Felix et al., 1999). In brief, a 96-well plate containing water and plant material (as described above) was incubated overnight at RT to remove ROS that had resulted from mechanical damage to the tissue during preparation. The next day, the water was replaced with 100 μL of fresh Milli-Q water containing 20 μg·mL-1 horseradish peroxidase (Sigma-Aldrich, Taufkirchen, Germany) and 20 μM L-012 (Wako Chemicals, Neuss, Germany). After a short incubation period (∼15 min), 100 μL of double-concentrated elicitor solutions were added to the wells. All elicitors were dissolved in Milli-Q water without additional treatment. Measurements of elicitor-triggered apoplastic ROS production were started immediately and performed continuously with an integration time of 450 ms in a TECAN SPARK 10 M multiwell plate reader (Männedorf, Switzerland). diff --git a/assays/ProteinPull-down/protocols/ProteinPull-downWithBiotinylatedLaminarin.md b/assays/ProteinPull-down/protocols/ProteinPull-downWithBiotinylatedLaminarin.md index e69de29..4e5882b 100644 --- a/assays/ProteinPull-down/protocols/ProteinPull-downWithBiotinylatedLaminarin.md +++ b/assays/ProteinPull-down/protocols/ProteinPull-downWithBiotinylatedLaminarin.md @@ -0,0 +1,2 @@ +## Protein pull-down with biotinylated laminarin +Barley leaves from 2-week-old plants were treated for 15 min with biotinylated laminarin, followed by vacuum infiltration for 2 min. Untreated laminarin and a biotinylated version of the bacterial elongation factor Tu peptide (elf18) were used as controls. The tissue was frozen in liquid nitrogen and ground to fine powder. Then, 10 mg·mL-1 of extraction buffer (10 mM MES, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% IGEPAL, proteinase inhibitor cocktail) were added to the powder. To avoid pH-dependent binding effects to the NeutrAvidin beads, two buffer conditions (pH 5.6 and pH 8.0) were used for all treatments. Samples were incubated rotating at 4 °C for 60 min and centrifuged at 10,645 g, 4 °C for 15 min. Supernatant was filtered to remove pieces, mixed with 50 μL of high-capacity NeutrAvidin agarose resin (Thermo Fisher Scientific, Schwerte, Germany), and incubated (inverting) at 4 °C for 3 h. The sample was briefly centrifuged at 60 g for 1 min. After discarding the supernatant, the beads were washed four times with 10 mL of wash buffer (10 mM MES [pH 5.6 or pH 8.0], 50 mM NaCl, 10 mM MgCl2, 0.5% IGEPAL). Proteins were eluted by boiling the beads with 50-70 μL of 2x SDS loading (including reducing agent) for 5 min. Proteins were separated by SDS-PAGE (NuPAGE; Invitrogen, Waltham, United States) after staining with Coomassie brilliant blue G-250, cut out for mass spectrometric analysis and digested with trypsin. \ No newline at end of file -- GitLab