Other than IPG‐DALT: 2‐DE variants

Abstract

Special problems require special solutions. That is why, despite the trend towards standardization of the procedures in proteomic investigations, a number of alternative protocols, based on 2-DE, have been devised with time and are applied to the resolution of proteins on which the performance of IPG-DALT is poor. The difficult-to-handle samples include high pI, high molecular size and high hydrophobicity proteins. The protocol variants entail changes in the gel media and/or the use of unusual additives (often surfactants or solvents). But also: Specific questions require specific answers. When the aim is not the resolution of individual polypeptide chains in a fully unfolded state but the analysis of higher-order structures (proteins assembled from subunits, complexes assembled from interacting proteins) the standard conditions under which IPG-DALT is performed turn inadequate. Electrophoresis is then performed either in first dimension or in both first dimension and second dimension under non-denaturing (native) and/or non-reducing conditions.

Two-dimensional gel electrophoresis in proteomics: A tutorial

“…Some review and/or practical papers are also of interest, either because they describe important caveats [36], or because they go deeper into more specialized topics, such as protein detection [37], non classical (i.e. without IEF) 2D gels [38] or variations to the basic electrophoretic protocols [39]. However, within the scope of this tutorial, we would like to show "the back side of the cards" in a stepwise fashion, and help the reader to understand the key parameters at play in 2D electrophoresis and the rationale underlying the protocols.…”

A decade of plant proteomics and mass spectrometry: Translation of technical advancements to food security and safety issues

“…In 2005, the first map‐based sequence of the annotated rice genome was also completed (International Rice Genome Sequencing Project, ). Entire genomes are now becoming available for some of the major crops, such as maize (Schnable et al, ), sorghum (Paterson et al, ), potato (Xu et al, ), tomato, soybean, domesticated apple (Velasco et al, ), or banana (D'Hont et al, ), as recently reviewed in (Feuillet et al, ; Miller, Eberinin, & Gianazza, ; Sonah et al, ) and at http://en.wikipedia.org/wiki/List_of_sequenced_eukaryotic_genomes. With the explosion in the amount of available data, it is increasingly difficult to provide a complete and updated picture of genome availability.…”

“…Classical 2-DGE is not suitable to analyze native proteins, protein complexes, and hydrophobic proteins. For an overview on the alternatives on classical 2-DGE, the reader is referred to Miller, Eberini, and Gianazza (2010). For a recent overview on the techniques used to analyze hydrophobic proteins in plants, readers are referred to Vertommen et al (2011b) and Kota and Goshe (2011).…”

Contamination of therapeutic human immunoglobulin preparations with apolipoprotein H (β2‐glycoprotein I)

“…In the present approach, we relied on 2DE under non‐reducing conditions as this allowed a better separation of the dominant protein IgG from proteinaceous impurities. As in other applications, this method has been able to visualize otherwise overlapping spot chains or patterns . In the present investigation, nonreducing 2DE proved useful for the study of protein impurities that differ between Ig products, as shown in Figs.…”