Abstract
Rhodopsin is a G-protein coupled receptor (GPCR) that mediates vision under dim light. Upon light exposure, rhodopsin is phosphorylated at multiple serine and threonine sites at its carboxyl-terminus by rhodopsin kinase (GRK1). This, in turn, reduces its ability to activate the visual G-protein transducin. Binding of light-activated, phosphorylated rhodopsin by arrestin (ARR1) fully terminates the catalytic activity of rhodopsin. Quantification of the levels of the differentially phosphorylated rhodopsin species provides definitive information about the role of phosphorylated rhodopsin in visual functions. Isoelectric Focusing (IEF) is a technique which is used to separate ampholytic components, such as proteins, based on their isoelectric point (pI). It is a useful technique used to distinguish protein isoforms and post-translational modifications such as phosphorylation, glycosylation, deamination, and acetylation, due to their effects on the protein's pI. Isoelectric Focusing can provide high resolution of differentially phosphorylated forms of a protein. Though other techniques such as kinase activity assays, phospho-specific antibodies, western blot, enzyme-linked immunosorbent assays (ELISA), radiolabeling and mass spectrometry are used to detect and quantify protein phosphorylation, IEF is a simple and cost-effective method to quantify rhodopsin phosphorylation, as it can readily detect individual phosphorylated forms. Here we provide a detailed protocol for determining phosphorylated rhodopsin species using the Isoelectric Focusing technique.