Kinase assays were carried out with a LabChip EZ Reader II system (PerkinElmer) at room temperature. The substrate used was a fluorescein-labelled peptide [5-FAM-FLAKSFGSPNRAYKK-CONH2] dissolved in 100 mM HEPES (pH 7.5), 0.003% (v/v) Brij-35, 0.004% (v/v) Tween-20, 10 mM MgCl2. Nek7WT (250 nM) or Nek7SRS (1 µM) enzyme was mixed in a 25 µl reaction volume, with 1.5 µM substrate peptide and the stated concentration of compound 51 before the addition of ATP was used to initiate the reaction. Measurements of substrate phosphorylation were taken every 2 min for 1 h. For the calculation for ATP Km, reaction mixes were prepared as described above with a 3-fold dilution series of ATP. Measurements of substrate phosphorylation were taken every 2 min for 1 h. All assays were carried out in duplicate. Data were plotted using Prism7 (http://www.graphpad.com ).
Labchip ez reader 2 system
Manufactured by PerkinElmer
Sourced in United States
The LabChip EZ Reader II system is a microfluidic analysis instrument designed for rapid and automated electrophoretic separation and detection of a variety of biological samples, including proteins, nucleic acids, and cells. The system utilizes miniaturized microfluidic chips to perform high-throughput analysis with minimal sample consumption.
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Lab products found in correlation
2 protocols using labchip ez reader 2 system
1
Kinase Assay of Nek7 Enzyme
2
Microfluidic Substrate Conversion Analysis
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The MMSA was carried out at room temperature using a LabChip EZ Reader II system from PerkinElmer (Waltham, USA) equipped with a 12-sipper chip in separation buffer. The reaction samples were sipped by a 12-sipper chip, and then the fluorescent product and substrate were separated under a screen pressure of -1.2 psi. In the step of detection, the fluorescent analytes were stimulated by a blue LED (450-490 nm) and detected by a CCD camera (515-550 nm).
The separation of peptide 15 was performed with the optimized conditions: upstream voltage = -500 V, downstream voltage = -2250 V, a screen pressure = -1.2 psi, base pressure = -0.5 psi. The separation buffer contained 100 mM HEPES, 1 mM EDTA, 0.015% Brij-35, 5% DMSO and 0.2% coating-3 reagent (PerkinElmer, Waltham, USA), pH 7.5. The substrate conversion rate was defined as the peak height of product divided by the sum of both substrate and product.
The separation of peptide 15 was performed with the optimized conditions: upstream voltage = -500 V, downstream voltage = -2250 V, a screen pressure = -1.2 psi, base pressure = -0.5 psi. The separation buffer contained 100 mM HEPES, 1 mM EDTA, 0.015% Brij-35, 5% DMSO and 0.2% coating-3 reagent (PerkinElmer, Waltham, USA), pH 7.5. The substrate conversion rate was defined as the peak height of product divided by the sum of both substrate and product.
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