Analysis of phosphorylation of unlabeled proteins

Phosphorylation of unlabeled proteins can be detected using immunological or enzymatic techniques. Anti-phosphotyrosine antibodies are used with immunoblots to detect tyrosine phosphorylation. This unit provides a protocol for detection of antibodies by 125I-labeled protein A or enhanced chemiluminescence (ECL). Detection of enzymatic dephosphorylation using a general or phosphoamino acid-specific phosphatase and subsequent SDS-PAGE mobility shift (or a change in activity) is also described.