In vitro Tyrosine Kinase Activity Assay


Materials
	Kinase buffer (125 mM NaCl; 48 mM MgCl₂; 50 mM HEPES (pH 7.5)
	4x SDS Buffer
	1 mM ATP
	Purified Kinase (e.g. Src or FAK)
	Kinase substrate (e.g. CEACAM3-cytoplasmic domain or FAK substrate)

0.2 μg/ml FAK-substrate or 25 ng/μl TwinStrep-Ceacam3-cytoplasmic domains
0.2 μg/ml hFAK KD (recombinant human FAK kinase domain) or 10 ng/μl Src
with or without (control) 100 μM ATP
in kinase buffer in a total volume of 30 μl/sample.
Kinase reactions are incubated at 37°C on an Eppendorf Thermomixer under constant mixing for 1h.
The kinase reaction is stopped by addition of 10 µl pre-heated 4 x SDS buffer and the samples are denatured for 5 min at 96 °C.
After centrifugation, the sample is loaded on a SDS-PAGE gel and processed for sequential Western Blotting with:
primary mouse α-pY (detecting tyrosine phosphorylated substrate)

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