Here are some highlights of a new paper describing a rapid method for analyzing changes in phosphorylation patterns due to knockdown of individual kinases in zebrafish embryos. The paper describes the use of a protein kinase substrate peptide array to detect changes in kinase activity among zebrafish morphants. I will not discuss many of the controls used in the original paper. For a detailed description of the experiments, see the original open-access paper at:
http://www.plosone.org/article/fetchArticle.action?articleURI=info%3Adoi%2F10.1371%2Fjournal.pone.0000581

Lemeer S, Jopling C, Naji F, Ruijtenbeek R, Slijper M, Heck AJ, den Hertog J. Protein-tyrosine kinase activity profiling in knock down zebrafish embryos. PLoS ONE. 2007 Jul 4;2:e581.

The activities of protein tyrosine kinases in lysates of one day old zebrafish embryos and in purified enzyme preparations were assayed. Twenty de-yolked zebrafish embryos were used for each pooled lysate preparation, providing enough lysate for at least five chip assays. For kinase knockdowns, translation-blocking Morpholinos were used which were designed to bind near the start codons of their mRNA targets. Morpholinos were microinjected into single-celled embryos at doses from 4 to 8 ng per embryo.

The purified SRC-family kinases Fyn and Yes have particular substrate peptides they preferentially phosphorylate. After knockdown of Fyn and Yes in embryos and treatment of the array with the embryo lysates, the array spots bearing those preferential Fyn/Yes substrates were less phosphorylated. Embryonic phenotypes of Fyn and Yes knockdowns are similar to Wnt11 knockdowns but differ from nacre/mitF phenotypes. Wnt11 knockdown or Fyn/Yes knockdown caused similar changes in the pattern of phosphorylation on the array; all of these differed from the phosphorylation pattern of nacre/mitF control knockdown embryos. Wild-type phosphorylation patterns could be partially recovered by coinjection of Morpholinos with their corresponding rescue mRNA for Fyn, Yes and Wnt11 knockdowns .

Peptide arrays on porous substrates allowed optimization of reaction kinetics by pumping samples up and down through the chip. Anti-phosphotyrosine antibodies with fluorescent labels were used for nonradioactive detection of phosphorylated peptides. Peptides were 15 amino acids in length. Zebrafish embryo lysates were pumped through the chips in a kinase reaction buffer containing ATP and anti-phosphotyrosine antibody. CCD images were taken of the chip every 3 minutes for 60 minutes, allowing kinetic analysis of fluorescence accumulation; analysis of the kinetics showed that none of the experimental spots had saturated with fluorescence by the end of the 60 minute assay.