Antibodies have been exploited as essential tools for the tracking of proteins of interest in biological samples to foster cell signalling research. Not only do they facilitate measurement of expression levels of proteins, but they can reveal alterations in their states of covalent modification, such as protein phosphorylation.

Kinexus has produced in-house over 1600 rabbit polyclonal antibodies against immunogenic peptides based on cell signalling proteins and tested these and over 3000 other commercial antibodies. Our internal testing has demonstrated that about three-quarters of commercially available antibodies are far more impotent or non-specific than often advertised. In fairness, cell signalling proteins are often expressed at levels that are magnitudes lower than structural proteins and metabolic enzymes. Moreover, the phosphorylated states of these proteins are often substoichiometric even when cells are prompted with agents that stimulate their phosphorylation. Due to the high degree of similarity in at least 250,000 known phosphosites, the opportunities for cross-reactivity are very high.

Another issue is that phosphosite-specific antibodies are often unavailable for interesting phosphosites in proteins that are highly conserved (hence they may be functionally important), are often highly phosphorylated (based on mass spectrometry studies) or likely to regulatory based on similarity in sequence with very related proteins for which the effects of phosphorylation have been described.