Protein Protocols Protein Protocols

Preparation of Eukaryotic Cells for 2-DE 159
21
Radiolabeling of Eukaryotic Cells and Subsequent
Preparation for 2-Dimensional Electrophoresis
Nick Bizios
1. Introduction
Two-dimensional polyacrylamide gel electrophoresis (2-DE) provides not only the
ability to resolve and quantify thousands of proteins, but it also gives those in research
and industry the ability to monitor in-process protein purification, quickly and easily
N. Bizios, unpublished date, (AGI Dermatics). 2-DE is also used to identify variability
in protein expression in a variety of cell lines (2,3). It is known that many parameters
and laboratory conditions can influence the resolution of proteins on 2-DE, such as the
pH range of carrier ampholytes used, the quality of reagents and equipment used, temperature,
voltage, and the skill of the researcher or technician.
One of the biggest obstacles one may encounter prior to performing 2-DE is the
proper documentation and validation of a Standard Operation Procedure (SOP). This
falls under the category of current Good Laboratory Practices (cGLPs) and or current
Good Manufacturing Practices (cGMPs). Without the proper documentation of reagents
used, such as Certificates of Analysis (readily supplied by the supplier’s Quality Control/Assurance
department), adherence to expiration dates of commercially prepared
reagents, implementation of expiration dates of reagents prepared in the laboratory,
and the adherence of SOPs (see Note 1) the ability of generating repeatable and consistent
results falls dramatically. However, the radiolabeling and preparation of eukaryotic
cells for 2-D may be considered paramount in obtaining consistent results. Without
proper radiolabeling and preparation, how would one properly find subsequent spots of
interest and perform quantitative analysis?
This chapter describes a general method for labeling methionine-containing proteins,
phosphorylation labeling, and subsequent lysate preparation for 2-DE that has
been modified from Garrels (3) and Garrels and Franza (4). The Jurkat T-lymphoblast
cell line is used as an example. A large number of protocols have been published for
the solubilization and sample preparation of eukaryotic cell lines and tissues for 2-DE.
One of the best sources for additional protocols is found at the Geneva University
Hospital’s Electrophoresis laboratory, which can be accessed at http://expasy.hcuge.ch/
ch2d/technical-info.html. (See also Chapters 19 and 20).
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
159