Protocol

Phosphoprotein Staining with the GelCode Phosphoprotein Staining Kit

This protocol was adapted from "Preparative 2D Gel Electrophoresis with Immobilized pH Gradients," Chapter 4 in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

The phosphorylation state of a protein has an important role in the regulation of a wide variety of cellular processes. As a result, there has been a great deal of interest in detecting phosphorylated proteins. The method presented here uses the GelCode phosphoprotein staining kit (Pierce Chemical Company). This method depends on the hydrolysis of the phosphoprotein phosphoester linkage using sodium hydroxide in the presence of calcium ions. The gel containing the newly formed insoluble calcium phosphate is then treated with ammonium molybdate in dilute nitric acid. The resultant insoluble nitrophospho-molybdate complex is stained with Methyl Green. After destaining, the phosphoproteins are colored green to green-blue. The detection limit is in the nanogram range, but depends on the degree of phosphorylation of the protein. This method will detect the phosphoproteins phosvitin and ß-casein in the 40-80 ng/band and 80-160 ng/band range, respectively. The method presented here is for staining minigels. Volumes will need to be increased for larger gels.