Purified AMPK (#171536, EMD Millipore, Billerica, MA) and/or ERK (#31152, Active Motif, Carlsbad, CA) were incubated with various recombinant substrates (0.5 μg) in kinase reaction buffer [HEPES (32 mM) pH 7.4, dithiothreitol (650 μM), Mg(CH3COO)2 (10 mM), BriJ-35 (0,012%), cold ATP (50 μM)] and 0.9–1.8 μCi of [γ-32P]ATP (Perkin Elmer, Waltham, MA) at 30 °C for 30 min. For AMPK, AMP (100 μM) was added to the reaction. Phosphorylation was detected by incorporation of radiolabeled [γ-32P]ATP.
Brij 35
Manufactured by PerkinElmer
Sourced in United States
BriJ-35 is a non-ionic surfactant commonly used in various laboratory applications. It is a polyoxyethylene(23)lauryl ether compound that can be utilized to solubilize and stabilize biological samples, proteins, and other compounds.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using brij 35
1
Protein Kinase Activity Assay
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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