Gel electrophoresis

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

Genetic Analysis of DNA Excreted in Urine: A New Approach for Detecting Specific Genomic DNA Sequences from Cells Dying in an Organism

Abstract: Background: Cell-free DNA from dying cells recently has been discovered in human blood plasma. In experiments performed on animals and humans, we examined whether this cell-free DNA can cross the kidney barrier and be used as a diagnostic tool. Methods: Mice received subcutaneous injections of either human Raji cells or purified 32P-labeled DNA. DNA was isolated from urine and analyzed by measurement of radioactivity, agarose gel electrophoresis, and PCR. In humans, the permeability of the kidney barrier to polymeric DNA was assessed by detection in urine of sequences that were different from an organism bulk nuclear DNA. Results: In the experiments on laboratory animals, we found that ∼0.06% of injected DNA was excreted into urine within 3 days in a polymeric form and that human-specific Alu sequences that passed through the kidneys could be amplified by PCR. In humans, male-specific sequences could be detected in the urine of females who had been transfused with male blood as well as in DNA isolated from urine of women pregnant with male fetuses. K- ras mutations were detected in the urine of patients with colon adenocarcinomas and pancreatic carcinomas. Conclusions: The data suggest that the kidney barrier in rodents and humans is permeable to DNA molecules large enough to be analyzed by standard genetic methodologies.

Alzheimer's disease: insolubility of partially purified paired helical filaments in sodium dodecyl sulfate and urea

Abstract: A method is described for the partial purification of the paired helical filaments that accumulate progressively in human neurons in Alzheimer's disease (senile dementia). Paired helical filaments have unusual solubility characteristics, including insolubility in sodium dodecyl sulfate, urea, reducing agent, and guanidine, which prevent analysis of their molecular composition by gel electrophoresis. The paired helical filaments appear to contain covalent bonds other than disulfide, which cross-link individual filaments into a rigid intracellular polymer. Thus, paired helical filaments appear to represent an example in neurons of an insoluble cross-linked protein. Covalently cross-linked protein polymers occur in lens senile cataracts and in terminally differentiated skin keratinocytes, suggesting that there may be a common mechanism for remodeling some structural proteins during cell aging.

Purification and physico-chemical characterization of rabbit tumor necrosis factor.

Abstract: Rabbit TNF has been purified 2000-fold by a series of salt precipitations, gel filtrations, ion exchange chromatography, and lectin affinity chromatography to a single species on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). TNF activity could be recovered from nondenaturing gel systems and has been shown to be an alpha-globulin with an isoelectric point of 5.1. The m.w. was estimated to be 68,000 d by SDS-PAGE, 55,000 by gel filtration, and 52,000 by glycerol gradient centrifugation. TNF activity was stable over the pH range of 6 to 10 and was relatively heat stable, not being inactivated at 70 degrees C for 1 hr. TNF activity was pronase sensitive, but relatively trypsin resistant. Neuraminidase and phospholipase C treatment did not destroy TNF activity. Partially purified TNF was still capable of eliciting hemorrhagic necrosis in susceptible tumors. Crude TNF serum had an interferon titer of 3000 U, whereas the partially purified sample had a titer of <30 U.