Protein kinases use ATP to phosphorylate certain amino acid residues (tyrosine, serine or threonine) on their substrates. In receptormediated signalling the phosphorylation forms an important secondary messenger system. Phosphorylation can be measured following radioactive 32P incorporation, which, however, involves the problems of radioactive work, safety and disposal. Eu-labelled anti-phosphotyrosine, -threonine and threonine-proline are available for the sensitive detection of phosphorylation levels on various substrates using time-resolved fluorometry and DELFIA as the detection technology.
Convenient collection of the substrates (peptides, polypeptides or proteins) on coated microtitration plates can be accomplished using streptavidin plates for biotinylated substrates or glutathione or anti-glutathione-S-transferase plates for substrates expressed with a GST tag.
1. In the assay, first incubate the substrate with enzyme (receptor), ATP and effectors.
2. After phosphorylation collect the phosphorylated products on coated plates. Incubate 1-2 hours and wash the plate two or three times with wash solution.
3. Add Eu-(or biotin-) labelled antibodies in DELFIA Assay Buffer (200 μl, 250 ng/ml) and incubate for 1-2 h. If performed in the indirect way, the secondary incubation with Eu-streptavidin takes 20-30 minutes.
4.Wash the plate six times with wash solution using the DELFIA Platewash (1296-024 or 026).
5. Add 200 μl of DELFIA Enhancement Solution, shake for 10 min with Plateshaker (1296-001/002 or 003/004) to guarantee a stable signal level and measure the plate with the Wallac time-resolved fluorometer.
Assays are currently routinely used in a number of drug discovery laboratories in the UK and USA. Braunwalder et al. Measurement of the protein tyrosine kinase activity of c-src using time-resolved fluorometry of europium chelates. Anal. Biochem. 238, 159-164 (1996).
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