Phos-tag beads as an immunoblotting enhancer for selective detection of phosphoproteins in cell lysates

The low specificity of anti-phosphoprotein antibodies is often a problem in immunoblotting analyses. We introduce a simple pretreatment procedure for cell lysates to give more specific detection of phosphoproteins in immunoblotting. Cellular phosphoproteins were preferentially trapped on Phos-tag agarose phosphate-affinity beads in a homemade spin-centrifuge microtube unit, and nonphosphorylated proteins were excluded in the filtrate. The phosphoprotein-bound beads suspended in a sample-loading dye solution were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western blotting. We demonstrated improved detection of phosphorylated Shc and mitogen-activated protein kinase isoforms in A431 cell lysates by this new technique.