Table 1. The KESTREL protocol.

The average time taken for each step is indicated in parentheses.

1.	Detect major proteins that are phosphorylated by the kinase of interest that are not phosphorylated by related kinases with similar specificities in vitro (days)
2.	Identify proteins by further purification and tryptic mass fingerprinting (a few weeks)
3.	Show protein is phosphorylated stoichiometrically and at a similar rate to an authentic physiological substrate (less than 1 day)
4.	Identify site of phosphorylation (a few days)
5.	Raise antibodies specific for the phosphorylation site (2–3 months)
6.	Show that the site is phosphorylated in vivo in response to stimuli that are known to activate the kinase of interest. Show that phosphorylation is prevented by drugs that inhibit the activity or activation of the kinase of interest (1 week–2 months)
7.	Show that phosphorylation does not occur in knockout mice lacking the kinase of interest (quick if mice are available!)
8.	Identify the function of the substrate. Then study the effect of phosphorylation on the function of the substrate, e.g. changes in activity, stability, subcellular location and interactions with other proteins (variable)