Kinase Assay
General Procedure:
1) Immunoprecipitate kinase
2) Incubate IP fraction with target substrate (ex. recombinant protein) and radio labelled ATP (the gamma phosphate of the ATP is radioactive P-32)
- If the kinase-substrate match is correct, the kinase will transfer the radioactive phosphate from ATP onto the substrate.
3) After incubation, resolve fraction of reaction mix on SDS-PAGE gel.
4) Dry gel, examine by autoradiography. Radioactive samples on the gel produce signal.
If the substrate was phosphorylated by the kinase, corresponding signal is produced on the autoradiogram. If the kinase was inactive or was unable to phosphorylate the chosen substrate, no signal is produced.
General Procedure:
1) Immunoprecipitate kinase
2) Incubate IP fraction with target substrate (ex. recombinant protein) and radio labelled ATP (the gamma phosphate of the ATP is radioactive P-32)
- If the kinase-substrate match is correct, the kinase will transfer the radioactive phosphate from ATP onto the substrate.
3) After incubation, resolve fraction of reaction mix on SDS-PAGE gel.
4) Dry gel, examine by autoradiography. Radioactive samples on the gel produce signal.
If the substrate was phosphorylated by the kinase, corresponding signal is produced on the autoradiogram. If the kinase was inactive or was unable to phosphorylate the chosen substrate, no signal is produced.