Revisiting electroblotting of immobilized pH gradient gels: A new protocol for studying post‐translational modification of proteins

Abstract

Currently, one of the most important techniques in proteome analysis is two-dimensional electrophoresis that is widely used for separation of thousands of different protein spots. Nevertheless, characterization of special aspects in protein patterns, e.g., separation of protein isoforms generated by post-translational modifications, requires individual detection methods, e.g., immunoblotting. Blotting of proteins after fractionation in immobilized pH gradients has always caused some problems. In this paper we present an optimized protocol for immunoblotting after isoelectric focusing using immobilized pH gradient (IPG) strips cast on Net-Fix as an internal support that is permeable to electric current. The focusing procedure can be carried out in commonly used IPG systems, e.g., the IPGphor by Amersham Biosciences, where electrically assisted rehydration can be performed. This may be of interest for many laboratories, because the same system as used for the first dimension of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is involved. As an example, we describe separation and detection of up to seven isoforms of recombinant erythropoietin beta using semidry blotting of IPG strips and visualization by chemiluminescence detection.

Identification and Characterization of Sulfolobus solfataricus P2 Proteome Using Multidimensional Liquid Phase Protein Separations

“…The presence of protein isoforms or post-translationally modified (PTM) proteins may give rise to different retention times during the chromatography, and therefore, the same protein will be identified from a number of fractions from the second dimension analysis. Protein isoforms and PTM proteins are also observed as different spots in 2DE, resulting in the same protein identification from multiple spots. , In addition, multiple proteins have been identified in a single broad peak obtained in the 2DLC analysis. These results suggest that protein resolution (peak capacity) still remains a problem using multidimensional liquid phase protein separations.…”

Immobilized pH gradients

“…Non‐ionic detergents readily saturate the blotting membrane 55; among them, only CHAPS may be effectively eluted from the focusing gel due to its high critical micellar concentration 56. For electrotransfer from IPGs, the plastic backing has to be disposed off 57, 58 or removed (using a Film Remover apparatus first developed by Pharmacia); but soft and thin IPG matrices may possibly swell, distort, even tear. To increase dimensional stability, polymerization with an internal support permeable to electric current was proposed 58, 59: GEL FIX™ (Serva) is made from polyester film chemically activated on both surfaces to provide covalent binding to the gel layer.…”

“…For electrotransfer from IPGs, the plastic backing has to be disposed off 57, 58 or removed (using a Film Remover apparatus first developed by Pharmacia); but soft and thin IPG matrices may possibly swell, distort, even tear. To increase dimensional stability, polymerization with an internal support permeable to electric current was proposed 58, 59: GEL FIX™ (Serva) is made from polyester film chemically activated on both surfaces to provide covalent binding to the gel layer. Moreover, the contact of the sticky IPG surface with nitrocellulose membranes is better avoided, pouring an agarose coating 58 or overlaying a cellulose acetate foil 60.…”

“…To increase dimensional stability, polymerization with an internal support permeable to electric current was proposed 58, 59: GEL FIX™ (Serva) is made from polyester film chemically activated on both surfaces to provide covalent binding to the gel layer. Moreover, the contact of the sticky IPG surface with nitrocellulose membranes is better avoided, pouring an agarose coating 58 or overlaying a cellulose acetate foil 60. As an alternative, diffusion blotting may be used 56, 61.…”

Application of microfluidic chip to realize controllable pH

“…Microfluidic analysis systems have many advantages over regular analysis systems such as high efficiency, low consumption of samples, low cost of chip fabrication and combinable multiple functions [3] . Since pH gradient plays an important role in separation and analysis of IEF and HPLC [4][5] , lots of pH gradient formation methods have been developed [6][7][8] .…”