Horizontal comparative fluorescence two‐dimensional gel electrophoresis for improved spot coordinate detection

Abstract

Vertical comparative 2D fluorescence gel electrophoresis (CoFGE) has recently been shown to increase the reproducibility of coordinate assignment for protein spots, in particular in singular experiments, which cannot be investigated using DIGE. The method applies a standardized marker grid formed by a set of purified proteins to the sample proteome in a conglomerate of 1DE, 2DE, and DIGE. Here, improvements are demonstrated by transferring CoFGE to horizontal 2DE. These include the elimination of the protein modification by residual acrylamide monomer unavoidable in vertical CoFGE, reduced buffer volumes, and highly efficient laboratory procedures. Spot patterns are well defined and can be easily analyzed using commercially available warping algorithms. With horizontal CoFGE also a correction for changes in pI was introduced using a third fluorescent dye. Horizontal CoFGE holds high promises in comparative proteomics.

Pyrylium‐based dye and charge tagging in proteomics

“…A number of advantages over traditional N ‐hydroxysuccinimidyl ester labels were listed such as the small size of the label, the rapid prestaining procedure, excellent stability and storability, and the color change indicating successful labeling (Py‐1: blue/nonfluorescent; product with primary amines: red/fluorescent) . We used LightningRed (LR) , the commercial product of Py‐1, for two‐dimensional comparative fluorescence gel electrophoresis, a technique we have developed for improved comparability of 2D‐PAGE of singular samples . In the course of optimization of experimental parameters, we became interested in the labeling mechanism, also driven by earlier experiences with unexpected side reactions during supposedly straightforward labeling , cross‐linking , or simply sample handling .…”

pI-Control in Comparative Fluorescence Gel Electrophoresis (CoFGE) using amphoteric azo dyes

“…Amphoteric azo dyes were used for the control of the first dimension (p I ) in horizontal Comparative two-dimensional Fluorescence Gel Electrophoresis (hCoFGE) [1] . CoFGE itself uses an internal reference grid formed by internal protein standards to correct for the gel-to-gel variation in the second dimension of 2D polyacrylamide GE improving protein spot coordinate assignment [2–4] .…”

Comparative Two-Dimensional Fluorescence Gel Electrophoresis

“…As long as it is performed with the same type of pI-strip and identical marker proteins, in silico matching of independent datasets is possible 4,7 . An exemplary dataset for the separation of Escherichia coli lysate, our test analyte 6,8 , is shown in Fig. 1.…”

“…CoFGE was rst developed for vertical 2D-PAGE using a 1D-PAGE comb for the generation of the marker wells 5,6 and it is still performed in this way by independent users 9,10 . However, we found it much more convenient to apply CoFGE to horizontal GE 8 as illustrated in Fig. 1, which has a number of advantages in itself.…”

“…For vertical GE, a stacking gel for the application of the references needs to be casted 5,6 , which is not necessary in horizontal GE, because ready-made gels are now commercially available, which include the wells for the reference grid (Mercator gels, Serva Electrophoresis). Alternatively, the slots for the marker wells can be manually punched out in regular 2D-gels using pipette tips as we did initially 8 . The CoFGE method as described above does not take into account the variability resulting from the rst dimension (IEF).…”