Teresa Goodman 1 Birte Schulenberg 1 Thomas H. Steinberg 1 Wayne F. Patton 2 1 Molecular Probes, Inc., Eugene, OR, USA 2 Perkin Elmer LAS, Boston, MA, USA Detection of phosphoproteins on electroblot membranes using a small-molecule organic fluorophore A new formulation of the small-molecule organic fluorophore, Pro-Q  Diamond dye, has been developed that permits rapid and simple detection of phosphoproteins di- rectly on polyvinylidene difluoride (PVDF) or nitrocellulose membranes (electroblots). Protein samples are first separated by electrophoresis and then electroblotted to mem- branes, stained and destained, in an analogous manner as typically performed with Amido Black or Ponceau S dye staining of total protein profiles. After staining, blots are imaged using any of a variety of laser-based gel scanners, xenon-arc lamp-based gel scanners or charge-coupled device (CCD) camera-based imaging devices equipped with UV trans- or epi-illumination. The uncomplicated and reliable staining protocol delivers results in as little as 1 h and the limit of detection for the stain is typi- cally 2–4 ng of phosphoprotein with a linear dynamic range of approximately 15-fold. Compared with traditional radiolabeling and antibody-based approaches, the new method offers significant advantages, including avoidance of radioactivity, no need for expensive antibodies, no requirement for blocking unoccupied sites on the mem- brane with protein or detergent solutions, no sequence context-specific binding to phosphorylated amino acid residues and the ability to analyze the native, steady-state phosphorylation of proteins obtained directly from tissue specimens or body fluids. Pro-Q Diamond dye binds directly and exclusively to the phosphate moiety, allowing it to detect the broadest spectrum of phosphorylated proteins possible. The stain binds noncovalently to phosphoproteins and is thus fully compatible with matrix- assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF- MS) or Edman sequencing. The blot stain is also compatible with standard colori- metric, fluorogenic, and chemiluminescent detection techniques employed in immu- noblotting. Keywords: Phosphopeptides / Phosphoproteins / Phosphoproteome / Signal transduction DOI 10.1002/elps.200406008 1 Introduction Phosphorylation is considered a fundamental covalent post-translational modification that regulates the func- tional status of proteins and peptides and in turn controls most cellular phenomena. Methods for determining the phosphorylation status of proteins and peptides are thus important with respect to the evaluation of diverse biolog- ical processes including carcinogenesis, signal trans- duction, apoptosis, cell division, cell motility, metabolism, gene regulation, and differentiation. Pro-Q Diamond dye is a unique fluorescence-based detection system for the specific and sensitive analysis of protein and peptide phosphorylation status in polyacrylamide gels, on micro- arrays and on polymeric beads, but to date has been unsuitable for the detection of phosphorylated proteins on polyvinylidene difluoride (PVDF) or nitrocellulose mem- branes due to high nonspecific background staining [1–4]. Hence, we have developed a new formulation of the fluor- ophore, referred to as Pro-Q Diamond phosphoprotein blot stain, which allows sensitive fluorescence detection of phosphoproteins electroblotted from 1-D and 2-D gels. Proteins are electroblotted onto membranes and stained with Pro-Q Diamond blot stain using a short and simple procedure. After staining, the blots are briefly destained and signal is detected using any of a variety of laser- based gel scanners, xenon-arc lamp-based gel scanners, or CCD camera-based systems equipped with UV epi- or transillumination. Pro-Q Diamond blot stain can quantify phosphoprotein loads ranging from 8 ng to 1 mg on a single blot. The blot stain binds noncovalently to phos- Correspondence: Teresa Goodman, Molecular Probes, Inc., 29851 Willow Creek Road, Eugene, OR 97402, USA E-mail: terrie.goodman@probes.com Fax: 1541-335-0504 Abbreviations: DDAO,9H-(1,3-dichloro-9,9-dimethylacridin-2- one-7-yl); PKA, protein kinase A Electrophoresis 2004, 25, 2533–2538 2533  2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim General