Immobilized pH gradient gels

Figure 11.6. Experimental arrangement for casting an immobilized pH gradient gel. A linear pH gradient is generated by mixing two solutions with initially equal volumes, where one solution is dense and the other is, and they are titrated to the extremes of the desired pH interval. The compensating rod in the reservoir is used as a stirrer after the addition of catalysts and for hydrostatically equilibrating the two solutions.

The high-resolution and separation stability afforded by isoelectric focusing on immobilized pH gradient gels has also been exploited in 2D electrophoresis methods, where IEF in the first dimension is followed by a second separation method performed in a perpendicular direction. 

For the first-dimension separation, a protein sample containing an appropriate cocktail of carrier ampholyte is applied to a standard immobilized pH gradient gel (IPG) strip and left overnight to rehydrate the strip. These strips are available in several narrow pH ranges for improved separation and are specially well suited for FFE-separated subfractions. Application of the appropriate electrical potential to the rehydrated strip results in separation of the proteins according to their intrinsic charge. 

Gorg, A., Obermaier, C., Boguth, G. and Weiss, W. (1 999) Recent developments in two-dimensional gel electrophoresis with immobilized pH gradients wide pH gradients up to pH 12, longer separation distances and simplified procedures. Electrophoresis 20, 712-717. 

D Lutomski, M Caron, J-D Cornillot, P Bourin, C Dupuy, M Pontet, D Bladier, R Joubert-Caron. Identification of different galectins by immunoblotting after two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients. Electrophoresis 17 600-606, 1996. 

Rabilloud, T., Valette, C., and Lawrence, J. J. (1994). Sample application by in-gel rehydration improves the resolution of two-dimensional electrophoresis with immobilized pH gradients in the first dimension. Electrophoresis 15, 1552-1558. 

The majority of proteomic investigations employ two-dimensional gel electrophoresis (2-D) with immobilized pH gradients (IPGs) to separate the proteins in a sample and combine this with mass spectrometry (MS) technologies to identify proteins. 

Hoving, S., Gerrits, B., Voshol, H., Muller, D., Roberts, R.C. and van Oostmm, J. (2002) Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients. Proteomics 2, 127-134. 

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