In:
ELECTROPHORESIS, Wiley, Vol. 33, No. 9-10 ( 2012-05), p. 1406-1410
Abstract:
The comparison of proteins separated on 2DE is difficult due to gel‐to‐gel variability. Here, a method named comparative fluorescence gel electrophoresis ( C o FGE ) is presented, which allows the generation of an artificial protein grid in parallel to the separation of an analytical sample on the same gel. Different fluorescent stains are used to distinguish sample and marker on the gel. The technology combines elements of 1DE and 2DE. Special gel combs with V ‐shaped wells are placed in a stacking gel above the p I strip. Proteins separated on the p I strip are electrophoresed at the same time as marker proteins (commercially available purified protein of different molecular weight) placed in V ‐wells. In that way, grids providing approximately 100 nodes as landmarks for the determination of protein spot coordinates are generated. Data analysis is possible with commercial 2DE software capable of warping. The method improves comparability of 2 DE protein gels, because they are generated in combination with regular in‐gel anchor points formed by protein standards. This was shown here for two comparative experiments with three gels each using E scherichia coli lysate. For a set of 47 well‐defined samples spots, the deviation of the coordinates was improved from 7% to less than 1% applying warping using the marker grid. Conclusively, as long as the same protein markers, the same size of p I ‐strips and the same technology are used, gel matching is reproducibly possible. This is an important advancement for projects involving comparison of 2 DE ‐gels produced over several years and in different laboratories.
Type of Medium:
Online Resource
ISSN:
0173-0835
,
1522-2683
DOI:
10.1002/elps.v33.9-10
DOI:
10.1002/elps.201200039
Language:
English
Publisher:
Wiley
Publication Date:
2012
detail.hit.zdb_id:
1475486-1
SSG:
12
