From 9d2fae8d902cf3608424a9ec02e966e680b9d79b Mon Sep 17 00:00:00 2001
From: Viktoria Petrova <vipet103@hhu.de>
Date: Thu, 6 Feb 2025 15:01:11 +0100
Subject: [PATCH] add protein and starch measurement assay and protocol

---
 assays/ProteinAndStarchMeasurements/README.md |   0
 .../dataset/.gitkeep                          |   0
 .../isa.assay.xlsx                            | Bin 0 -> 6903 bytes
 .../protocols/.gitkeep                        |   0
 .../ProteinAndStarchMeasurementsProtocol.md   |   5 +++++
 assays/TitratableAcidity/isa.assay.xlsx       | Bin 17253 -> 17253 bytes
 6 files changed, 5 insertions(+)
 create mode 100644 assays/ProteinAndStarchMeasurements/README.md
 create mode 100644 assays/ProteinAndStarchMeasurements/dataset/.gitkeep
 create mode 100644 assays/ProteinAndStarchMeasurements/isa.assay.xlsx
 create mode 100644 assays/ProteinAndStarchMeasurements/protocols/.gitkeep
 create mode 100644 assays/ProteinAndStarchMeasurements/protocols/ProteinAndStarchMeasurementsProtocol.md

diff --git a/assays/ProteinAndStarchMeasurements/README.md b/assays/ProteinAndStarchMeasurements/README.md
new file mode 100644
index 00000000..e69de29b
diff --git a/assays/ProteinAndStarchMeasurements/dataset/.gitkeep b/assays/ProteinAndStarchMeasurements/dataset/.gitkeep
new file mode 100644
index 00000000..e69de29b
diff --git a/assays/ProteinAndStarchMeasurements/isa.assay.xlsx b/assays/ProteinAndStarchMeasurements/isa.assay.xlsx
new file mode 100644
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diff --git a/assays/ProteinAndStarchMeasurements/protocols/.gitkeep b/assays/ProteinAndStarchMeasurements/protocols/.gitkeep
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index 00000000..e69de29b
diff --git a/assays/ProteinAndStarchMeasurements/protocols/ProteinAndStarchMeasurementsProtocol.md b/assays/ProteinAndStarchMeasurements/protocols/ProteinAndStarchMeasurementsProtocol.md
new file mode 100644
index 00000000..5890e9ef
--- /dev/null
+++ b/assays/ProteinAndStarchMeasurements/protocols/ProteinAndStarchMeasurementsProtocol.md
@@ -0,0 +1,5 @@
+## Protein and Starch Measurements
+
+Approximately 25 mg of ground leaf material was used for extraction in 80% (v/v) ethanol with 10 mM 2-(N-morpholino)ethanesulfonic acid (pH 6). Extraction was performed three times with 500 µl extraction buffer while shaking for 1 hour at 90 °C. After each extraction step, samples were centrifuged at 12,000 g for 10 min and the supernatant was collected and pooled with previous fractions. From each sample, two extracts were prepared and stored at –20 °C until analysed. The protein concentration was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).
+
+Pellets obtained during protein extraction were gelatinized with 300 µl 0.2 M NaOH at 90 °C for 40 minutes. After adjustment to pH 5–6, 200 µl hydrolysis buffer was added [20 mM acetate buffer, pH 4.8; 0.5 U α-amylase (10102814001, Roche), and 4 U amyloglucosidase (A7095, Roche)] and the mixture was incubated overnight at 37 °C. Glucose resulting from starch degradation was measured enzymatically: 10 µl of digest was added to 100 mM HEPES-NaOH (pH 7.5) with 10 mM MgCl2, 1 mM NADP+, 2 mM ATP, 0.1 U glucose-6-phosphate dehydrogenase (G6PDHII-RO, Roche), and 4 U hexokinase (11426362001, Roche). The final reaction volume was 200 µl and the increase of absorbance at 340 nm was monitored.
\ No newline at end of file
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