Abstract
DIGE (differential in-gel electrophoresis) is a modified version of the widely used 2-D gel electrophoresis (2-DE) for separation of complex protein samples. Two extracts to be compared are differentially labeled using fluorescent cyanine dyes and then separated together by 2-DE. An internal standard labeled using a third dye is included. This approach avoids the pitfalls of gel distortions frequently observed in the standard procedure, which hamper the subsequent gel image analysis. Inclusion of an internal standard improves the quantitative evaluation of the protein patterns. Using the advantages of the DIGE approach, impact of minor temperature differences during cold stress treatment could be quantitatively monitored. We will describe the application of DIGE to monitor the impact of cold stress on the proteome pattern of Arabidopsis. In addition to the separation of proteins, we will also outline how plant growth is performed. Finally, we will also give protocols how proteins of interest can be identified by MALDI-TOF- as well as ESI-MS/MS.
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Schlesier B, Mock H-P (2006) Protein isolation and second-dimension electrophoretic separation. In: Salinas J, Sanchez-Serrano J (eds) Arabidopsis protocols. Humana Press, Totowa, NJ, pp 381–391
Damerval C, Devienne D, Zivy M, Thiellement H (1986) Technical improvements in two-dimensional electrophoresis increase the level of genetic-variation detected in wheat-seedling proteins. Electrophoresis 7:52–54
Laemmli UK (1970) Cleavage of structural proteins during assembly of head of bacteriophage T4. Nature 227:680–685
Kang DH, Gho YS, Suh MK, Kang CH (2002) Highly sensitive and fast protein detection with coomassie brilliant blue in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. B Kor Chem Soc 23:1511–1512
Amme S, Matros A, Schlesier B, Mock H-P (2006) Proteome analysis of cold stress response in Arabidopsis thaliana using DIGE-technology. J Exp Bot 57:1537–1546
GE Healthcare (2009) 2-D Quant Kit. Little Chalfont: GE Healthcare. https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314729545976/litdoc28954714AE_20110830215136.pdf
GE Healthcare (2005) Ettan DIGE system user manual, 18-1173-17 Edition AB. Uppsala: GE Healthcare. https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314774443672/litdoc18117317AB_20110831091933.pdf
GE Healthcare (2010) 2-D Electrophoresis. Principles and methods. Little Chalfont: GE Healthcare. https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1335426794335/litdoc80642960_20140202231631.pdf
Branham WS, Melvin CD, Han T, Desal VG, Moland CL, Scully AT, Fuscoe JC (2007) Elimination of laboratory ozone leads to a dramatic improvement in the reproducibility of microarray gene expression measurements. BMC Biotechnol 7:8
Acknowledgment
Support of the proteomic research performed by the authors obtained from IPK Gatersleben and from BMBF research grants (FKZ 0315047A & FKZ 0315699) is gratefully acknowledged.
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Döll, S., Lippmann, R., Mock, HP. (2014). Proteomic Approaches to Identify Cold-Regulated Soluble Proteins. In: Hincha, D., Zuther, E. (eds) Plant Cold Acclimation. Methods in Molecular Biology, vol 1166. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0844-8_12
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DOI: https://doi.org/10.1007/978-1-4939-0844-8_12
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-0844-8
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