MAP Kinase Signaling Protocols (Methods in Molecular Biology by Rony Seger

By Rony Seger

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Wash the gel carefully four times (15 min each at room temperature) with 5% TCA + 1% NaPPi. If the gel is still very radioactive, continue washing overnight. 9. Dry the gel and subject to autoradiography. Bands should appear where kinases are present and caused phosphorylation of the MBP copolymerized in the gel. 10. The rate of phosphorylation of up to nine protein kinases can be enhanced by different treatment, although not all of them can be seen under all conditions. In Fig. 3, phosphorylation by six protein kinases is enhanced in Chinese hamster ovary (CHO) cells overexpressing ErbB2 on stimulation with EGF.

However, because the separation of various protein kinases is not always complete, and because its laborious nature, the method is not widely in use and is not described here. Another method that is used for the detection of novel protein kinases is the in-gel kinase assay. This technique involves copolymerization of a given substrate in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) of the samples of interest on the copolymerized gel, and in-gel phosphorylation of the embedded substrate in the presence of [a32P]-ATP.

3. Detection of MAPK activities by in-gel kinase assay. 1% FCS, 18 h) and then treated with EGF (50 ng/mL) for the indicated times. 5. Arrows indicate the calculated molecular weight of the bands. 8. Wash the gel carefully four times (15 min each at room temperature) with 5% TCA + 1% NaPPi. If the gel is still very radioactive, continue washing overnight. 9. Dry the gel and subject to autoradiography. Bands should appear where kinases are present and caused phosphorylation of the MBP copolymerized in the gel.

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