Abstract
Identifying signaling molecules that participate in pathways leading from receptor activation to changes in gene expression in cells is the fundamental focus of many research endeavors. For example, neurotrophins interacting with their trk receptors mediate a number of cellular effects, including cell differentiation, neurite outgrowth, and neuronal survival (1–4). This results from specific protein-protein interactions in different signaling pathways. The primary aim of many research efforts is to elucidate these signaling pathways by identifying the proteins that participate in them. The most common method for isolating protein-protein interactions is by coimmunoprecipitation of protein complexes from cell lysates, where an appropriate antibody is available. Isolated proteins are separated using polyacrylamide gel electrophoresis and then transferred from the gel to a membrane support and identified using immunoblotting. These techniques have been well documented (see refs. 5–8). The major drawback of this technique is its inability to find novel proteins. Currently, the best technique available for identifying novel proteins is by chemical or enzymatic fragmentation of isolated proteins in a gel matrix following one- or two-dimensional electrophoresis or after electrotransfer onto a suitable membrane (9,10). In most cases, reverse-phase high-performance liquid chromatography (RP-HPCL) is used to separate the cleavage fragments.
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References
Batistatou, A., Volonté, C., and Greene, L. A. (1992) Nerve growth factor employs multiple pathways to induce primary response genes in PC 12 cells. Mol. Biol. Cell. 3, 363–371.
Volonté, C., Angelastro, J. M., and Greene, L. A. (1993) Association of protein kinases ERK1 and ERK2 with p75 nerve growth factor receptors. J. Biol. Chem. 268, 21,410–21,415.
Heumann, R. (1994) Neurotrophin signalling. Curr. Opin. Neurobiol. 4, 668–679.
Kaplan, D. R. and Stephens, R. M. (1994) Neurotrophin signal transduction by the Trk receptor. J. Neurobiol. 25, 1404–1417.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of the’ bacteriophage T4. Nature 227, 680–685.
Midgley, C. A. and Lane, D. P. (1993) Looking for protein-protein interactions, in Cellular Interactions in Development: A Practical Approach (Hartley, D. A., ed.), Oxford University Press, Oxford, pp. 129–151.
Bradd, S. J. and Dunn, M. J. (1993) Analysis of membrane proteins by western blotting and enhanced chemiluminescence. Methods Mol. Biol. 19, 211–218.
Dunn, M. J. and Bradd, S. J. (1993) Separation and analysis of membrane proteins by SDS-polyacrylamide gel electrophoresis. Methods Mol. Biol. 19, 203–210.
Rosenfeld, J., Capdevielle, J., Guillemot, J. C., and Ferrara, P. (1992) In-gel digestion of proteins for internal sequence analysis after one-or two-dimensional gel electrophoresis. Anal. Biochem. 203, 173–179.
Hellman, U., Wernstedt, C., Gonez, J., and Heldin, C. H. (1995) Improvement of an “In-Gel” digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal. Biochem. 224, 451–455.
Fenselau, C. (1997) MALDI MS and strategies for protein analysis. Anal. Chem. 69, 661A–665A.
Merril, C. R., Goldman, D., Sedman, S. A., and Ebert, M. H. (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211, 1437,1438.
Merril, C. R., Goldman, D., and Van Keuren, M. L. (1984) Gel protein stains: silver stain. Methods Enzymol. 104, 441–447.
Celis, J. E., Rasmussen, H. H., Leffers, H., Madsen, P., Honore, B., Gesser, B., et al. (1991) Human cellular protein patterns and their link to genome DNA sequence data: usefulness of two-dimensional gel electrophoresis and microsequencing. FASEB J. 5, 2200–2208.
James, P., Quadroni, M., Carafoli, E., and Gonnet, G. (1994) Protein identification in DNA databases by peptide mass fingerprinting. Protein Sci. 3, 1347–1350.
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© 2001 Humana Press Inc.
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Bartlett, S.E. (2001). Identifying Novel Proteins in Nervous Tissue Using Microsequencing Techniques. In: Rush, R.A. (eds) Neurotrophin Protocols. Methods in Molecular Biology™, vol 169. Humana Press. https://doi.org/10.1385/1-59259-060-8:43
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DOI: https://doi.org/10.1385/1-59259-060-8:43
Publisher Name: Humana Press
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