Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

The overall goal of the following experiment is to label the proteins of interest with radioactive phosphates in order to quantify the proteins, cellular phosphorylation levels. This is achieved by culturing cells in the presence of phosphorus 32 to radioactively labeled the phosphate modifications of proteins expressed in the cells. As a second step labeled cells are lysed and lysates are incubated with a affinity beads for immuno purification of the proteins to be analyzed.

Next samples of labeled and purified proteins are run on sodium do desal sulfate, poly acrylamide gel electrophoresis and blotted to poly olaine fluoride membranes. In order to detect radioactive phosphate incorporation and protein levels, results are obtained that show leucine rich repeat kinases one and two, abbreviated L rrk one and L rrk two are phosphorylated in cells to comparable levels based on auto radiography and immuno detection. A main advantage of the metabolic labeling technique compared to other techniques such as phospho, immuno blotting, are that this technique allows the determination of total cellular phosphorylation levels of proteins including for proteins for which no phospho antibodies are available.