Fyn Activity Assay Protocol

Fyn activity assay was performed with Universal Tyrosine Kinase Assay kit (Takara Bio Inc, Shiga, Japan) as described previously [27 (link)]. Briefly, homogenates were prepared and protein concentration was determined by BCA method. Samples (0.5-1 mg) were incubated with 4 μg of Fyn rabbit polyclonal antibody (Santa Cruz Biotechnology) coupled with Catch and Release^® v2.0 Reversible Immunoprecipitation System (EMD Millipore). Samples were washed three times with NP-40 lysis buffer and once with the kinase reaction buffer. Immuno-complexes were diluted 2.5 fold and 40 μl samples and 10 μl ATP solution were incubated in an ELISA plate provided. Measurement of the kinase activity was performed according to the manufacturer's protocol.

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Yamada E., Okada S., Bastie C.C., Vatish M., Nakajima Y., Shibusawa R., Ozawa A., Pessin J.E, & Yamada M. (2016). Fyn phosphorylates AMPK to inhibit AMPK activity and AMP-dependent activation of autophagy. Oncotarget, 7(46), 74612-74629.

Publication 2016

Universal Tyrosine Kinase Assay kit Catch and Release® v2.0 Reversible Immunoprecipitation System

Antibody Assay Buffer Elisa Immunoprecipitation Kinase Np 40 Protein Rabbit Tyrosine kinase

Corresponding Organization : Gunma University

Other organizations : University of Warwick, University of Oxford, Albert Einstein College of Medicine

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Variable analysis

independent variables

Amount of Fyn rabbit polyclonal antibody (4 μg)

dependent variables

Fyn kinase activity

control variables

Protein concentration (0.5-1 mg)
Incubation time and conditions
Wash steps (3 times with NP-40 lysis buffer and once with kinase reaction buffer)
Volume of samples (40 μl) and ATP solution (10 μl)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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