Results and reliability of protein quantification for two‐dimensional gel electrophoresis strongly depend on the type of protein sample and the method employed

Abstract

We investigated the effects of tissue samples taken from rat brain on the reliability of three protein quantification kits: the Bradford assay, the 2-D Quant Kit, and the EZQ Protein Quantitation Kit. All three assays measured significantly smaller amounts of protein after extraction than the reference values before extraction. Only small effects were seen in homogenates, but very pronounced differences in membrane-enriched and highly lipophilic subcellular fractions. Researchers should evaluate which method of protein quantification is best qualified for their specific experimental design.

Proteomic Analysis Reveals Age-related Changes in Tendon Matrix Composition, with Age- and Injury-specific Matrix Fragmentation

“…Assessment of the normalization factor used by Progenesis during relative quantification revealed higher normalization factors for old samples even though a fixed amount of protein (based on Bradford assay results) was loaded. This suggests that the Bradford assay may have provided an incorrect estimation of sample protein content, as reported previously (47), but the reasons for this are unclear. Taken together, these results suggest that in aged tendon protein extractability was reduced, suggesting that the matrix in aged samples is more resistant to degradation, with more proteins remaining trapped within the insoluble portion of the matrix.…”

The utility of functional interaction and cluster analysis in CNS proteomics

“…A number of articles exemplify this Eravci et al, 2008;Weist et al, 2008). The first step in the analysis of most proteomic studies, as described and illustrated above, is to simply detect candidate proteins that significantly change between control and disease tissue.…”

Multi-dimensional liquid chromatography in proteomics—A review

“…Although 2-DGE is a powerful technique for protein separation, it has a number of severe limitations [15,16]. The process is difficult to automate, labor intensive, slow, and prone to contamination with unresolved proteins.…”