add protein and starch measurement assay and protocol

assays/ProteinAndStarchMeasurements/protocols/ProteinAndStarchMeasurementsProtocol.md

+## 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.
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