Protein detection in gels using fixation.

This unit has been recently updated to include information on preformulated gel stains as well as new protocols for Sypro Ruby and silver staining, and gel imaging methodology. Other previously published protocols are also provided for both rapid and acid-based Coomassie blue staining, and alternate methods for silver staining (i.e.

Higher dimensional (Hi-D) separation strategies dramatically improve the potential for cancer biomarker detection in serum and plasma.

The plasma proteome has a wide dynamic range of protein concentrations and is dominated by a few highly abundant proteins. Discovery of novel cancer biomarkers using proteomics is particularly challenging because specific biomarkers are expected to be low abundance proteins with normal blood concentrations of low nanograms per milliliter or less. Conventional, one- and two-dimensional proteomic methods including 2D PAGE, 2D DIGE, LC-MS/MS, and LC/LC-MS/MS do not have the capacity to consistently detect many proteins in this range.

A novel four-dimensional strategy combining protein and peptide separation methods enables detection of low-abundance proteins in human plasma and serum proteomes.

A novel strategy, termed protein array pixelation, is described for comprehensive profiling of human plasma and serum proteomes. This strategy consists of three sequential high-resolution protein prefractionation methods (major protein depletion, solution isoelectrofocusing, and 1-DE) followed by nanocapillary RP tryptic peptide separation prior to MS/MS analysis. The analysis generates a 2-D protein array where each pixel in the array contains a group of proteins with known pI and molecular weight range.

Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma.

Systematic detection of low-abundance proteins in human blood that may be putative disease biomarkers is complicated by an extremely wide range of protein abundances. Hence, depletion of major proteins is one potential strategy for enhancing detection sensitivity in serum or plasma. This study compared a recently commercialized HPLC column containing antibodies to six of the most abundant blood proteins ("Top-6 depletion") with either older Cibacron blue/Protein A or G depletion methods or no depletion.

Enhanced analysis of human breast cancer proteomes using micro-scale solution isoelectrofocusing combined with high resolution 1-D and 2-D gels.

Current methods for quantitatively comparing proteomes (protein profiling) have inadequate resolution and dynamic range for complex proteomes such as those from mammalian cells or tissues. More extensive profiling of complex proteomes would be obtained if the proteomes could be reproducibly divided into a moderate number of well-separated pools. But the utility of any prefractionation is dependent upon the resolution obtained because extensive cross contamination of many proteins among different pools would make quantitative comparisons impractical.