ESO wikiadmin: Created page with "{| class="wikitable" |+ !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) |} === Procedure === ---- * 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)..."

2025-02-27T12:08:37Z

Created page with "{| class="wikitable" |+ !Materials ! |- | |Kinase buffer (125 mM NaCl; 48 mM MgCl<sub>2</sub>; 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) |} === Procedure === ---- * 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)..."

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{| class="wikitable"
|+
!Materials
!
|-
|
|Kinase buffer (125 mM NaCl; 48 mM MgCl<sub>2</sub>; 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)
|}

=== Procedure ===
----

* 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)

In vitro Tyrosine Kinase Activity Assay - Revision history