Before phosphorylation, recombinant SYNJ2BP (WT or S21A, 0.5 µg) was dephosphorylated using 1 µl of CIP (NEB) in 9 µl 1× CutSmart buffer. The reactions were incubated at 37 °C for 1 h and stopped by 1× PhosStop (Roche). In vitro phosphorylation of dephosphorylated recombinant SYNJ2BP (0.5 µg in 10 µl) was performed in kinase reaction buffer (8 mM MOPS/NaOH, pH 7 and 200 µM EDTA) with 500 µM ATP (Serva), 200 µM AMP (Serva) and active recombinant AMPK (16 ng) (Sigma-Aldrich, 14-840) in a total volume of 30 µl. Alternatively, neuronal lysates were prepared from cells by Dounce homogenization in lysis buffer (20 mM Tris/HCl, pH 7.2, 30 mM NaCl, 10 mM MgCl2, 10% glycerol, 1 mM EDTA, 200 µM PMSF, protease inhibitor cocktail (Roche) and PhosStop (Roche)) and cleared by centrifugation at 2,300g for 1 min at 4 °C. The supernatant was collected and immediately used for in vitro phosphorylation assays or snap-frozen. Then, 0.5 µg SYNJ2BP was mixed with 25 µl cytosolic extract supplemented with 500 µM ATP in a final volume of 40 µl. The samples were incubated at 30 °C for 2 h (shaking). The reactions were stopped by addition of Laemmli sample buffer at 95 °C for 5 min.
Adenosine triphosphate (atp)
Manufactured by Serva Electrophoresis
Sourced in United States
ATP is a laboratory equipment used for electrophoresis applications. It functions as a power supply, providing the necessary electrical current and voltage for the separation and analysis of biomolecules, such as proteins and nucleic acids, within a gel matrix.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using adenosine triphosphate (atp)
1
In Vitro Phosphorylation of SYNJ2BP
2
Neuro-2A-Luc-Cell Luciferase Assay
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In order to test the luciferase expression of the stably transduced Neuro-2A-Luc-cell line, 4,000 cells were seeded into three 24-well-plates. After 4, 7 and 11 days the luminescence was measured. For this purpose, the culture medium was removed and a 100µl luciferase assay solution, containing D-Luciferin (25 mM, Sigma Aldrich, St. Loius, MO, USA), Tricine (200 mM, MERCK, Darmstadt, Germany), MgSO 4 (50 mM, MERCK, Darmstadt, Germany), MgCO 3 (1.07 mM, VWR, Radnor, PA, USA), DTT (500 mM, Thermo-Scientific, Waltham, MA, USA ), ATP (25 mM, SERVA, Heidelberg, Germany) and EDTA (10 mM, FLUKA, St.Gallen, Switzerland) for luminescence development and Triton X (1%, Sigma, St. Louis, MO,USA ) for cell lysis was added and mixed properly. The luminescence was measured 30 minutes after the addition by a TECAN F200 infinite microplate reader in triplicates (Männedorf, Switzerland). The acute toxicity study was performed to evaluate the tolerability of targeted nanoplexes at different dosing (15 µg, 30 µg and 55 µg total per animal, 2 mice per group). The compound was injected once i.v. (50 µl injection volume). The animals were observed for two weeks and the body weight was measured during the study.
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