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Additional resources for Biological Concepts and Techniques in Toxicology: An Integrated Approach
A related approach avoids Cy dyes but incorporates the multiplexing strategy by means of multiple, repetitive staining of individual gels to detect all proteins in the 2-D gel pattern, along with phosphoproteins (20) and glycoproteins (21). This methodology enables parallel determination of altered glycosylation and phosphorylation patterns and protein expression level changes without running three gels. Large-Scale, Highly Parallel 2-DE. An alternative to multiplexing samples in 2-DE by DIGE is found in highly parallel 2-DE separations.
Abbreviation: 2-DE, two-dimensional electrophoresis. Source: From Ref. 24. ’’ Its use (or misuse) here is intended as a point of emphasis with respect to the vast dynamic range of protein expression and our desire to ‘‘sharply focus’’ on and accurately analyze expression of the least abundantly expressed protein alongside the most abundant protein, and everything in between. To improve analytical depth for 2-DE, sIEF provides perhaps the best and most attractive remedy. This approach subdivides a complex protein mixture into well-resolved fractions based on the pI of each protein in the mixture.
Finally, combining the power of DIGE and rodent airway epithelial cell isolation, Wheelock et al. (19) improved the applicability of 2-DE-based proteomics to respiratory toxicology by demonstrating the enrichment (by 36%) of epithelial cell-speciﬁc proteins and resolving 2365 detectable spots. A related approach avoids Cy dyes but incorporates the multiplexing strategy by means of multiple, repetitive staining of individual gels to detect all proteins in the 2-D gel pattern, along with phosphoproteins (20) and glycoproteins (21).