Detection and quantitation of changes in the state of phosphorylation of specific proteins is of great utility in the quest to establish the function of a given protein and the consequences of its reversible phosphorylation. Two methods commonly used to measure protein phosphorylation and dephosphorylation in cell preparations employ prelabeling with 32P or back phosphorylation. These methods continue to be very effective and have advantages for many test systems, but they do have several practical and theoretical limitations (Nestler and Greengard, 1984). Based in large part on the successful use of short synthetic peptides to produce epitope-targeted antibodies (Lerner, 1982;Sutcliffe et al., 1983), an immunochemical approach became an attractive alternative for detecting changes in the state of phosphorylation of specific proteins at a specific site. The use of phosphorylation state-specific antibodies takes advantage of the sensitivity and selectivity afforded by immunochemical methodology, combined with relatively simple preparation and potentially broad applications. The first report of phosphorylation-dependent antibodies appeared in 1981, when polyclonal antibodies that could detect phosphotyrosine-containing proteins were produced by immunization with benzyl phosphonate conjugated to keyhole limpet hemocyanin (KLH) (Ross et al., 1981). Shortly thereafter, Nairn and colleagues reported the production of serum antibodies that distinguished between the phospho- and dephospho-forms of G-substrate, a protein localized to cerebellar Purkinje cells and phosphorylated by cGMP-dependent protein kinase (Nairn et al., 1982). A synthetic heptapeptide, Arg-Lys-Asp-Thr-Pro-Ala-Leu, corresponding to a repeated sequence surrounding two phosphorylated threonyl residues in the intact protein, served as antigen. Rabbit antisera against a peptide-KLH conjugate were specific for the dephospho-form of G-substrate. Phospho-specific antibodies were prepared by immunization of rabbits with the purified phosphoprotein, phosphorylated in vitro to a stoichiometry of 2 mol/mol with cGMP-dependent protein kinase. Despite this initial success, other attempts in our laboratory to produce phospho-specific polyclonal antisera by immunization with the phospho-form of intact proteins were not very successful, probably because of two significant factors. First, many phosphorylated proteins are believed to undergo rapid dephosphorylation during immunization, regardless of the route of injection, leading to the loss of the desired phospho-epitope. Second, holoproteins generally contain multiple immunogenic epitopes; this decreases the probability that clonal dominance for a phospho-specific epitope will be obtained.
Taking a more direct approach utilizing phosphorylated and unphosphorylated forms of synthetic phosphopeptides, we developed a general protocol for the production of phosphorylation state-specific antibodies for substrates with established site(s) of phosphorylation (Czernik et al., 1991)). In early stages of our development of this methodology, phosphopeptides were routinely prepared by enzymatic phosphorylation (Czernik et al., 1991). Although this approach remains perfectly valid today, the preparation of synthetic phosphopeptides using Fmoc derivatives of phosphoamino acids has become the state-of-the-art (Czernik et al., 1995;Czernik et al., 1996). Likewise, we have examined the use of both polyclonal and monoclonal techniques for antibody production. Given the high success rate that we and others have obtained with the polyclonal technique, it has become the method of choice, because it is an easier and less costly method for the average laboratory. However, when appropriate, this approach can be readily adapted for monoclonal antibody production.
- Czernik AJ, Girault J-A, Nairn AC, Chen J, Snyder G, Kebabian J, Greengard P (1991) Production of phosphorylation state-specific antibodies. Methods Enzymol 201: 264-283.
- Czernik AJ, Mathers J, Mische SM (1997) Phosphorylation state-specific antibodies. Neuromethods: Regulatory Protein Modification: Techniques & Protocols 30: 219-250.
- Czernik AJ, Mathers J, Tsou K, Greengard P, Mische SM (1995) Phosphorylation state-specific antibodies: preparation and applications. Neuroprotocols 6: 56-61.
- Lerner, R. A. Tapping the immunological repertoire to produce antibodies of predetermined specificity. Nature 299, 593-596. 1982.
- Nairn AC, Detre JA, Casnellie JE, Greengard P (1982) Serum antibodies that distinguish between the phospho- and dephospho-forms of a phosphoprotein. Nature (Lond ) 299: 734-736.
- Nestler, E. J. and Greengard, P. Protein Phosphorylation in the Nervous System. Nestler and Greengard. Protein Phosphorylation in the Nervous System. , 255-299. 1984. New York, Wiley.
- Sutcliffe JG, Shinnick TM, Green N, Lerner RA (1983) Antibodies that react with predetermined sites on proteins. Science 219: 660-666.