Functional characterization of two-dimensional gel-separated proteins using sequential staining

Jian Wu, Nataliya J. Lenchik, Michael J. Pabst, Solomon S. Solomon, James Shull, Ivan Gerling

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

Proteins separated by two-dimensional (2-D) gel electrophoresis can be visualized using various protein staining methods. This is followed by downstream procedures, such as image analysis, gel spot cutting, protein digestion, and mass spectrometry (MS), to characterize protein expression profiles within cells, tissues, organisms, or body fluids. Characterizing specific post-translational modifications on proteins using MS of peptide fragments is difficult and labor-intensive. Recently, specific staining methods have been developed and merged into the 2-D gel platform so that not only general protein patterns but also patterns of phosphorylated and glycosylated proteins can be obtained. We used the new Pro-Q™ Diamond phosphoprotein dye technology for the fluorescent detection of phosphoproteins directly in 2-D gels of mouse leukocyte proteins, and Pro-Q™ Emerald 488 glycoprotein dye to detect glycoproteins. These two fluorescent stains are compatible with general protein stains, such as SYPRO Ruby stain. We devised a sequential procedure using Pro-Q™ Diamond (phosphoprotein), followed by Pro-Q™ Emerald 488 (glycoprotein), followed by SYPRO Ruby stain (general protein stain), and finally silver stain for total protein profile. This multiple staining of the proteins in a single gel provided parallel determination of protein expression and preliminary characterization of post-translational modifications of proteins in individual spots on 2-D gels. Although this method does not provide the same degree of certainty as traditional MS methods of characterizing post-translational modifications, it is much simpler, faster, and does not require sophisticated equipment and expertise in MS.

Original languageEnglish (US)
Pages (from-to)225-237
Number of pages13
JournalElectrophoresis
Volume26
Issue number1
DOIs
StatePublished - Jan 1 2005

Fingerprint

Gels
Staining and Labeling
Coloring Agents
Proteins
Phosphoproteins
Mass Spectrometry
Post Translational Protein Processing
Mass spectrometry
Glycoproteins
Diamond
Ruby
Peptide Fragments
Electrophoresis, Gel, Two-Dimensional
Body Fluids
Fluorescent Dyes
Silver
Proteolysis
Body fluids
Leukocytes
Electrophoresis

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

Cite this

Functional characterization of two-dimensional gel-separated proteins using sequential staining. / Wu, Jian; Lenchik, Nataliya J.; Pabst, Michael J.; Solomon, Solomon S.; Shull, James; Gerling, Ivan.

In: Electrophoresis, Vol. 26, No. 1, 01.01.2005, p. 225-237.

Research output: Contribution to journalArticle

Wu, Jian ; Lenchik, Nataliya J. ; Pabst, Michael J. ; Solomon, Solomon S. ; Shull, James ; Gerling, Ivan. / Functional characterization of two-dimensional gel-separated proteins using sequential staining. In: Electrophoresis. 2005 ; Vol. 26, No. 1. pp. 225-237.
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