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: 15738
评审: Tie Liu
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参见作者原研究论文
The authors used this protocol in:
Dec 2012
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 )
- SimplyBlueTM SafeStain protein stain (Life Technologies, Invitrogen™, catalog number: LC-6065 )
- Precision PlusTM 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
- EttanTM IPGphoreTM 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
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
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.
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分类
生物化学 > 蛋白质 > 电泳
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