Abstract
The genome information combined with data derived from modern mass spectrometry enables us to determine the identity of a protein once it is isolated from a complex mixture. Two-dimensional gel electrophoresis established more than four decades ago servesĀ as a powerful protocol to isolate many proteins at once for such protein analysis. In the first two decades, the original procedure to use a glass tube-based IEF had been commonly used. Since an IEF in glass tubes is rather difficult to maneuver, a new method to use an IEF on a thin agarose slab backed by a plastic film (IPG Dry Strip) had been invented and is now widely used. In this chapter, we describe a protocol that uses a glass tube-based IEF because the capacity of protein loading and resolving power of this type of classic two-dimensional gel is still indispensable for many applications, not only for protein identification but also for protocols that are benefited by larger amounts of materials, i.e., analysis of posttranslational modification of proteins such as phosphorylation, methylation, glycosylation, and others.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Matsumoto H, Komori N (2000) Ocular proteomics: cataloging photoreceptor proteins by two-dimensional gel electrophoresis and mass spectrometry. Methods Enzymol 316:492ā511
Matsumoto H, Kurono S, Matsumoto M, Komori N (2005) Mass spectrometry of biomolecules in proteomics. In: Meyers RA (ed) Encyclopedia of molecular and cell biology and molecular medicine. Wiley-VCH Verlag GmbH, Germany, pp 557ā585
Kenrick KG, Margolis J (1970) Isoelectric focusing and gradient gel electrophoresis: a two-dimensional technique. Anal Biochem 33:204ā207
Klose J (1975) Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik 26:231ā243
OāFarrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007ā4021
Scheele GA (1975) Two-dimensional gel analysis of soluble proteins. Characterization of guinea pig exocrine pancreatic proteins. J Biol Chem 250:5375ā5385
Ames GF, Nikaido K (1976) Two-dimensional gel electrophoresis of membrane proteins. Biochemistry 15:616ā623
Miyazaki K, Hagiwara H, Yokota M, Kakuno T, Horio T (1978) In: Ui N, Horio T (eds) Isoelectric focusing and isotachophoresis. Kyoritsu Shuppan, Tokyo, p. 183. [In Japanese]
Matsumoto H, Pak WL (1984) Light-induced phosphorylation of retina-specific polypeptides of Drosophila in vivo. Science 223:184ā186
Matsumoto H, Kurien B, Takagi Y, Kahn ES, Kinumi T, Komori N, Yamada T, Hayashi F, Isono K, Pak WL, Jackson KW, Tobin SL (1994) Phosrestin I undergoes the earliest light-induced phosphorylation by a calcium/calmodulin-dependent protein kinase in Drosophila photoreceptors. Neuron 12:997ā1010
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680ā685
Haniu H, Komori N, Takemori N, Singh A, Ash JD, Matsumoto H (2006) Proteomic trajectory mapping of biological transformation: Application to developmental mouse retina. Proteomics 6:3251ā3261
Komori N, Takemori N, Kim HK, Kurono S, Singh A, Hwang S-H, Foreman RD, Chung K, Chung JM, Matsumoto H (2007) Proteomics study of neuropathic and non-neuropathic dorsal root ganglia: Altered protein regulation following segmental spinal nerve ligation injury. Physiol Genomics 29:215ā230
Shitama T, Hayashi H, Noge S, Uchio E, Oshima K, Haniu H, Takemori N, Komori N, Matsumoto H (2008) Proteome profiling of vitreoretinal diseases by cluster analysis. Proteomics Clin Appl. 2:1265ā1280
Kinumi T, Jackson KW, Ohashi M, Tobin SL, Matsumoto H (1997) The phosphorylation site and desmethionyl N-terminus of Drosophila phosrestin I in vivo determined by mass spectrometric analysis of proteins separated on two-dimensional gel electrophoresis. Eur Mass Spectrom 3:367ā378
Takemori N, Komori N, Matsumoto H (2006) Highly sensitive multistage mass spectrometry enables small scale analysis of protein glycosylation from two-dimensional polyacrylamide gel. Electrophoresis 27:1394ā1406
Takemori N, Komori N, Thompson J Jr, Yamamoto M-T, Matsumoto H (2007) Novel eye-specific calmodulin methylation characterized by protein mapping in Drosophila melanogaster. Proteomics 7:2651ā2658
Takemori N, Komori N, Matsumoto H (2009) Chapter 150: MS analysis of protein glycosylation. In: Walker JM (ed) The protein protocols handbook, 3rd edn. Humana Press, New York, pp 1381ā1388
Steinberg RA, Coffino P (1979) Two-dimensional gel analysis of cyclic AMP effects in cultured S49 mouse lymphoma cells: protein modifications, inductions and repressions. Cell 18:719ā733
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
Ā© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Matsumoto, H., Haniu, H., Kurien, B.T., Komori, N. (2019). Two-Dimensional Gel Electrophoresis by Glass Tube-Based IEF and SDS-PAGE. In: Kurien, B., Scofield, R. (eds) Electrophoretic Separation of Proteins. Methods in Molecular Biology, vol 1855. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8793-1_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8793-1_11
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8792-4
Online ISBN: 978-1-4939-8793-1
eBook Packages: Springer Protocols