Gel electrophoresis

About: Gel electrophoresis is a research topic. Over the lifetime, 26026 publications have been published within this topic receiving 1113565 citations.

Application of laser capture microdissection combined with two-dimensional electrophoresis for the discovery of differentially regulated proteins in pancreatic ductal adenocarcinoma.

Abstract: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal of all the common malignancies and markers for early detection or targets for treatment of this disease are urgently required. The disease is characterised by a strong stromal response, with cancer cells usually representing a relatively small proportion of the cells in the tumor mass. We therefore performed laser capture microdissection (LCM) to enrich for both normal and malignant pancreatic ductal epithelial cells. Proteins extracted from these cells were then separated by two-dimensional gel electrophoresis (2-DE). The limited amounts of protein in the LCM procured samples necessitated the detection of 2-DE resolved proteins by silver staining. Consequently, loading equivalent amounts of protein onto gels was essential. However, we found that conventional means of measuring total protein in the samples were not sufficiently accurate. We therefore adopted a strategy in which the samples were first separated by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis, stained with silver stain and subjected to densitometry. Evaluation of the staining intensity was then used to normalise the samples. We found that the protein profiles from undissected normal pancreas and LCM-acquired non-malignant ductal epithelial cells from the same tissue block were different, underpinning the value of LCM in our analysis. The comparisons of protein profiles from nonmalignant and malignant ductal epithelial cells revealed nine protein spots that were consistently differentially regulated. Five of these proteins showed increased expression in tumor cells while four showed diminished expression in these cells. One of the proteins displaying enhanced expression in tumor cells was identified as the calcium-binding protein, S100A6. To determine the incidence of S100A6 overexpression in pancreatic cancer, we carried out immunohistochemical analysis on sections from a pancreas cancer tissue array containing 174 duplicate normal and malignant pancreatic tissue samples, from 46 pancreas cancer patients. Normal pancreatic ductal epithelia were either devoid of detectable S100A6 or showed weak expression only. Moderately or poorly differentiated tumors, by contrast, showed a higher incidence and a higher level of S100A6 expression. These observations indicate that the combination of LCM with 2-DE provides an effective strategy to discover proteins that are differentially expressed in PDAC.

Purification and characterisation of a membrane-bound substance-P-degrading enzyme from human brain.

Abstract: A membrane-bound enzyme which degrades substance P (an undecapeptide) has been purified from human brain. The properties of this enzyme suggest that it may be involved in the physiological inactivation of the peptide by neural tissues. Enzyme activity was extracted from a membrane fraction of human diencephalon with a non-ionic detergent, Brij 35, and activity was monitored by measuring the disappearance of added substance P using radioimmunoassay, bioassay or radiochemical assay. The enzyme was purified about 1000-fold by chromatography on DEAE-cellulose, hydroxyapatite and Sephadex gel filtration columns. To identify the cleavage sites in substance P, the peptide was incubated with the purified enzyme and the breakdown products were separated by reverse-phase high-performance liquid chromatography and identified by amino acid analysis. The results suggested that the enzyme preparation was functionally homogeneous and it cleaved substance P between Gln6–Phe7, Phe7–Phe8 and Phe8-Gly9, with no exopeptidase action. The enzyme had a pH optimum in the range 7–9 and was strongly inhibited by metal-chelating agents, but not affected by most other peptidase inhibitors; it can thus be classified as a neutral metallo-endopeptidase. The enzyme was thermolabile and had a molecular weight of 40000–50000 as estimated by gel filtration, density-gradient ultracentrifugation and sodium dodecylsulphate gel electrophoresis. The highly purified substance-P-degrading enzyme could be distinguished from previously described peptidases for which substance P is a substrate. An important feature was that substance P was the preferred substrate among various other neuropeptides tested.