Fig. 1

Experimental setup of Kinase-Associated Phosphoisoform Assay. The concept is based on the differential phosphoisoform distribution of candidate substrates transiently expressed with or without co-expression of activated kinases. Full-length cDNAs of protein kinase(s) and candidate substrate(s) are cloned into plant expression vectors as translational fusion constructs. Use of fusion proteins containing commonly used epitopes also circumvents the need of specific antibodies. Candidate substrates are transfected into protoplasts, where intracellular phosphorylation can occur. Following an appropriate incubation period the protoplasts are harvested, lysed and the protein extracts are loaded into capillaries. Isoelectric focusing takes place in a pH gradient within the capillaries. Finally, separated proteins are immobilised to the capillary surface, and detected by chemiluminescent enzyme-coupled antibodies, specific against the epitopes fused to the substrate proteins