Antibodies to Phospho-
Proteins & Phosphopeptides

Phosphorylation is a fundamental posttranslational modification that control the biological activity of a of a large number biomolecules including enzymes, growth and differentiation factors, receptors and oncogenes. Phosphorylation is controlled by two classes of enzymes, the kinases which put the phophate onto the substrate, and the phosphatases, which remove the phosphate form the substrate. Kinases have been pursued heavily as drug targets by the pharmaceutical industry, and recent pharmaceutical breakthroughs include two kinase inhibitors, Gleevec™ and Iressa™. Screening for kinase inhibitors and many other research and biotechnological applications often require antibodies to phosphorylated proteins or peptides, whose binding is dependent on the phosphorylation state of the antigen, i.e. the antibody will bind to the phosphorylated form only but not recognize the unphosphorylated form. For phosphopeptides, antisera specific for the phosphorylated form can often be generated by simple immunizations of rabbits with phosphopeptide conjugated to an immunogenic carrier. Phosphoproteins are usually targeted with monoclonal antibodies that specifically recognize the phosphorylated epitope or another conformational epitope that depends on phosphorylation. In the case of phosphoproteins and phsophopeptides that are phosphorylated on a tyrosine residue, monoclonal antibodies have become available commercially, that recognize the phosphorylated tyrosine independently of its microenvironment and are useful to probe for unknown substrates of pharmaceutically important protein tyrosine kinases.

Antibody Solutions has custom-made a number of antibodies to phosphopeptides and phosphoproteins. The example below illustrates the differential actitvities of rabbit antisera that we made against a specific phosphopeptide. It shows that these sera clearly distinguish between the phosphorylated and non-phosphorylated form of the peptide. Such antibodies have numerous applications including the use in homogenous phase high throughput screening (SPA-assay).

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