Preparation and application of antibodies to phosphoamino acid sequences

In this review, we describe methods to generate and characterize sequence-specific phosphoamino acid antibodies. Several of the early contributions regarding the utility of such antibodies are summarized. Three antiphosphopeptide antibodies derived from sequences of the Bcr protein are described. They are anti-Bcr pSer-354, anti-Bcr pTyr-328, and anti-Bcr pTyr-360. These anti-Bcr phosphopeptide antibodies are directed toward phosphorylated sequences encoded by the first exon of the BCR gene, which is the critical portion of the Bcr sequence present in the Bcr-Abl oncoprotein. Using these antibodies, we established/confirmed the in vivo phosphorylation of Ser-354, Tyr-328, and Tyr-360 in Bcr and Bcr-Abl proteins. The cross-reactivity of these antibodies, which is a common problem with antipeptide antibodies, was also investigated and discussed.