Acsii aqueous counting scintillant

The SnRK1 activity assays were performed in a final volume of 25 μl. The reaction mixture was prepared with 40 mM HEPES (pH 7.5), 5 mM MgCl₂, 200 μM ATP containing 12.5 kBq (γ‐³²P) ATP (Perkin Elmer), 4 mM DTT, 1× phosphatase inhibitor, 1× protease inhibitor cocktail (Sigma), 200 μM AMARA peptide (AMARAASAAALARRR), PHT1;8 peptide (DMQRVMSRSHISRRR), PHR1 peptide (PVHRSGSRDLTRRR), PHO1 peptide (GLIKTYSSINMRRR), and NP‐GAPDH peptide (PVLRINSVEEGIRRR). The assay was started by addition of the sample containing protein kinase complexes. After 6 min, 15 μl of the reaction mixture was transferred to a square (2 × 2 cm) piece of phosphocellulose paper (Whatman P81), which was then immersed in 1% (v/v) phosphoric acid. The papers were rinsed twice with phosphoric acid, followed by acetone. The membranes were air‐dried, and the incorporation of ³²P was quantified by the addition of scintillation liquid (ACSII aqueous counting scintillant, Amersham) and measurement in a Beckman scintillation counter.

The SnRK1 activity assays were conducted in a total volume of 25 μl at 30°C. The reaction mixture for each assay contained 40 mM HEPES, pH 7.5, 5 mM MgCl₂, 200 μM ATP containing 12.5 kBq (γ−³³P) ATP (PerkinElmer, MA USA), 200 μM of the AMARA peptide (AMARAASAAALARRR), 4 mM DTT, 0.5 μM okadaic acid and 1× protease inhibitor cocktail (Sigma, Mexico). In some activity assays, 0.4 mg of A/A or 0.5 mg of starch granules was included in the reaction mixture. The assay was then initiated by the addition of the extract containing the protein kinase. After 6 min, a 15 μL aliquot of the reaction mixture was transferred to a square (2 × 2 cm) piece of phosphocellulose paper (Whatman P81, Whatman), which was immediately immersed in 1% (v/v) phosphoric acid. The papers were washed three times with phosphoric acid, followed by acetone. The incorporation of ³³P was quantified by liquid scintillation counting (ACSII Aqueous Counting Scintillant, Amersham) in a Beckman Scintillation Counter.