Immunofluorescence (IF) takes advantage of biological and physical mechanisms to identify proteins in cell or tissue samples, exploiting the specificity of antibodies and stimulated fluorescence light emission. Here, we describe an immunofluorescence staining method for the identification of histidine phosphorylated proteins that uses neutral/alkaline conditions and targeted reagents to overcome the chemical lability of histidine phosphorylation. This method describes how 1- and 3-phosphohistidine (pHis) monoclonal antibodies can be used to reveal the localization of proteins containing these elusive phosphoramidate bonds in cells. Standard procedures and materials for IF staining with adherent and nonadherent cells are described.
Keywords: Antibody; Fluorescence; Histidine phosphorylation; Immunofluorescence; Immunostaining; Microscopy; Phosphohistidine.