IEF-2DE 分析和蛋白质鉴定

Xia Wu; Steven J.  Clough; Steven C.  Huber; Man-Ho Oh

doi:10.21769/BioProtoc.924

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Peer-reviewed

IEF-2DE Analysis and Protein Identification

IEF-2DE 分析和蛋白质鉴定

发布: 2013年10月05日第3卷第19期 DOI: 10.21769/BioProtoc.924 浏览次数: 15632

评审: Tie Liu

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参见作者原研究论文

The authors used this protocol in:

Cover of Plant Physiology, featuring study using the protocol.

Dec 2012

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Abstract

Isoelectric focusing followed by SDS-PAGE (IEF-2DE) separates proteins in a two-dimensional matrix of protein pI (Protein Isoelectric Point) and molecular weight (MW). The technique is particularly useful to distinguish protein isoforms (Radwan et al., 2012) and proteins that contain post-translational modifications such as phosphorylation (Oh et al., 2012) and lysine acetylation (Wu et al., 2011). Proteins that are separated by IEF-2DE can be identified by immunoblot analysis using sequence-specific antibodies or by mass spectrometry.

Materials and Reagents

Protein extracts
Phenol (TE-saturated pH 7.9) (Sigma-Aldrich, catalog number: P4557 )
0.1 M ammonium acetate (dissolved in methanol)
Tris-base (General Electric Company, catalog number: 17-1321-01 )
Glycine
Urea (Life Technologies, Invitrogen™, catalog number: ZU10001 )
Thiourea (Life Technologies, Invitrogen™, catalog number: ZT10002 )
CHAPS (Life Technologies, Invitrogen™, catalog number: ZC10003 )
DTT (General Electric Company, catalog number: 17-1318-01 )
Bromophenol blue (General Electric Company, catalog number: 17-1329-01 )
Glycerol (General Electric Company, catalog number: 17-1325-01 )
SDS (General Electric Company, catalog number: 17-1313-01 )
Iodoacetamide (General Electric Company)
100% Ethanol (HPLC grade)
Water (HPLC grade)
IPG buffer (General Electric Company, catalog number: 17-6000-87 )
IPG strips (General Electric Company, catalog number: 17-6000-14 )
Immobiline DryStrip Cover Fluid (General Electric Company, catalog number: 17-1335-01 )
Bradford assay dye reagent (Bio-Rad Laboratories, catalog number: 500-0006 )
SimplyBlue^TM SafeStain protein stain (Life Technologies, Invitrogen™, catalog number: LC-6065 )
Precision Plus^TM All Blue Protein Standards (Bio-Rad Laboratories, catalog number: 161-0373 )
IEF sample buffer (see Recipes)
SDS equilibration buffer (see Recipes)
10x Tank buffer (see Recipes)
Agarose overlay solution (General Electric Company, catalog number: 17-0554-01 ) (see Recipes)

Equipment

1.5 ml microfuge tubes
15 ml tubes
Ettan^TM IPGphore^TM Isoelectric Focusing System (General Electric Company, Model: 80-6414-02 )
Large Format 1-D Electrophoresis Systems (Bio-Rad Laboratories)
Semi-dry transfer system (Bio-Rad Laboratories)
Mass spectrometers (such as MALDI-TOF or tandem mass spectrometer)

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Wu, X., Clough, S. J., Huber, S. C. and Oh, M. (2013). IEF-2DE Analysis and Protein Identification. Bio-protocol 3(19): e924. DOI: 10.21769/BioProtoc.924.
Radwan, O., Wu, X., Govindarajulu, M., Libault, M., Neece, D. J., Oh, M. H., Berg, R. H., Stacey, G., Taylor, C. G., Huber, S. C. and Clough, S. J. (2012). 14-3-3 proteins SGF14c and SGF14l play critical roles during soybean nodulation. Plant Physiol 160(4): 2125-2136.