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Multiplexed Proteomics Technology for the Fluorescence Detection of Phosphorylation and Protein Expression Levels Using Pro-Q® Diamond and SYPRO® Ruby Dyes

  • Birte Schulenberg
  • Terrie Goodman
  • Thomas H. Steinberg
  • Wayne F. Patton
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

The reversible phosphorylation of serine, threonine, and tyrosine residues is arguably one of the most important covalent posttranslational modifications regulating the functional status of proteins in eukaryotic organisms. Tools and techniques for determining the phosphorylation status of proteins and peptides thus play a prominent role in the investigation of diverse biological phenomena, including signal transduction, cell division, cell motility, apoptosis, metabolism, differentiation, gene regulation, and carcinogenesis. Typically, cells or isolated proteins are labeled with 32 P or 33 P prior to gel electrophoresis using protein kinases (1). The phosphoproteins are then usually detected by autoradiography, using film or storage phosphor imaging screens. In vitro radiolabeling provides a measure only of the phosphate groups attached during the actual labeling. No information is provided with respect to the physiological phosphorylation status of the proteins (2). Radiolabeling phosphoproteins also presents safety issues associated with handling the material and contamination of instrumentation (3).

Keywords

Sodium Dodecyl Sulfate Barium Carbonate Include Signal Transduction Sodium Dodecyl Sulfate Sample Buffer Alkaline Phosphatase Treatment 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • Birte Schulenberg
    • 1
  • Terrie Goodman
    • 1
  • Thomas H. Steinberg
    • 1
  • Wayne F. Patton
    • 1
  1. 1.Molecular Probes, Inc.Eugene

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