Detecting Protein Phosphorylation

Protein phosphorylation is a key regulatory signaling event that can activate or deactivate protein function and regulate critical biochemical pathways. Phosphospecific antibodies represent research windows into those pathways: They allow researchers to determine where and when a protein is phosphorylated and how these modifications affect protein function and interactions. Typically, a researcher probes a Western blot with phosphospecific antibodies to obtain a profile of protein phosphoryla

Wendy Gloffke
Sep 29, 2002

Protein phosphorylation is a key regulatory signaling event that can activate or deactivate protein function and regulate critical biochemical pathways. Phosphospecific antibodies represent research windows into those pathways: They allow researchers to determine where and when a protein is phosphorylated and how these modifications affect protein function and interactions.

Typically, a researcher probes a Western blot with phosphospecific antibodies to obtain a profile of protein phosphorylation. But phosphoELISA™ kits from Biosource International of Camarillo, Calif., allow scientists to detect and quantify specific phosphorylated proteins using a 96-well plate instead. The assay works much like an immunoprecipitation experiment. A plate is coated with an antibody that pulls out all of the specific protein from a cell lysate or tissue homogenate. Next, a phosphospecific antibody detects the phosphorylated residue on the protein of interest. Finally, a secondary antibody labeled with horseradish peroxidase is added for subsequent colorimetric detection. To normalize phosphorylation levels,...

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