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sodium dodecyl sulfate-stable oligomers in cell culture

Time:2015/11/24 5:50:35

An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels

The use of 3,3′,5,5′-tetramethylbenzidine-HOas a stain for the peroxidase activity of cytochrome-450 (or cytochrome-450 in sodium dodecyl sulfate polyacrylamide gels is described in this report. This reagent can be used to detect very low levels of heme-associated peroxidase activity. The blue-stained bands on polyacrylamide gels are distinet, and the color is stable. The stained gels can be photographed or scanned at 690 nm because the gel background remains clear. The stain is easily removed from the gels to permit subsequent protein staining. Staining first for peroxidase activity has no effect on the subsequent protein staining profile. The peroxidase activity of cytochrome-450 (or cytochrome-420) in immunoprecipitates in Ouchterlony double diffusion plates can also be detected using this reagent.

Aggregation of secreted amyloid-protein into sodium dodecyl sulfate-stable oligomers in cell culture

Filamentous aggregates of the 40-42-residue amyloid β-protein (Aβ) accumulate progressively in the limbic and cerebral cortex in Alzheimer's disease, where they are intimately associated with neuronal and glial cytopathology. Attempts to model this cytotoxicity in vitro using synthetic peptides have shown that monomeric Aβ is relatively inert, whereas aggregated Aβ reproducibly exerts a variety of neurotoxic effects. The processes that mediate the conversion of monomeric Aβ into a toxic aggregated state are thus of great interest. Previous studies of this conversion have employed high concentrations (10 -10 M ) of synthetic Aβ peptides under nonbiological conditions. We report here the detection of small amounts (<10 M ) of SDS-stable Aβ oligomers in the culture media of Chinese hamster ovary cells expressing endogenous or transfected amyloid β-protein precursor genes. The identity of these oligomers (primarily dimers and trimers) was established by immunoprecipitation with a panel of Aβ antibodies, by electrophoretic comigration with synthetic Aβ oligomers, and by amino acid sequencing. The oligomeric Aβ species comprised 10-20% of the total immunoprecipitable Aβ in these cultures. A truncated Aβ species beginning at Arg 5 was enriched in the oligomers, suggesting that amino-terminal heterogeneity can influence Aβ oligomerization in this system. Addition of Congo red (10 μ M ) during metabolic labeling of the cells led to increased monomeric and decreased oligomeric Aβ. The ability to detect and quantitate oligomers of secreted Aβ peptides in cell culture should facilitate dynamic studies of the critical process of initial Aβ aggregation under physiological conditions.