Universal tyrosine kinase assay kit

Kinase activity of FAK was measured with a nonradioactive isotope solid-phase enzyme-linked immunosorbent assay (ELISA) kit using the poly(Glu, Tyr) as a substrate (Universal Tyrosine Kinase Assay kit; Takara Biomedical Technology, Beijing, hina). FAK was purified from cells by immunoprecipitation with a mouse monoclonal anti-focal adhesion kinase (FAK) (B-8; sc-271195) antibody (Santa Cruz Biotechnology). Immunoprecipitates were subjected to the in vitro kinase assay as per the manufacturer’s instructions (Takara Biomedical Technology). Each experiment was repeated for three times in duplicates.

A Universal Tyrosine Kinase Assay Kit (Takara, Melbourne, Australia) was used to determine the enzymatic activity of rSmFGFRA at different concentrations (100 ng/μl - 0.78 ng/μl) in the presence or absence of 5 μM and 10 μM BIBF 1120 as described (14 (link), 36 (link)), following the manufacturer’s instructions. DMSO (0.1%) was served as negative control. The activity of rSmFGFRA was determined by comparing its absorbance with that of control protein tyrosine kinase (PTK), supplied with the kit, according to the manufacturer’s instructions. Technical duplicates were performed and the experiment was repeated twice.

NPM-ALK tyrosine kinase activity was measured using the Universal Tyrosine Kinase Assay Kit (Takara Bio, Pittsburg, PA). Briefly, 2 × 10⁶ cells were immunoprecipitated using standard protocol and ALK antibody (Dako) overnight at 4°C. Samples were spun and resuspended (including beads) in Kinase Reacting Solution and plated in triplicates in the protein tyrosine kinase substrate immobilized microplate (provided in the kit). ATP-2Na was added to each well in order to start tyrosine phosphorylation and incubated at 37°C for 30 min. The sample solution was then aspirated, and the wells were washed 4 times. Subsequently, samples were blocked with blocking solution for 30 min at 37°C, followed by addition of 50 μL of anti-phosphotyrosine (PY20)-horseradish peroxidase (HRP) solution for 30 min at 37°C. Finally, samples were washed and 100 μL of the HRP substrate TMB was added into each well for 15 min. The reaction was stopped by adding 100 μL of stop solution and absorbance was measured at 450 nm.

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.