Search results for "Density gradient ultracentrifugation"
showing 8 items of 8 documents
Effects on lipoprotein subclasses of combined expression of human hepatic lipase and human apoB in transgenic rabbits
2003
Objective— The effects of combined expression of human hepatic lipase (HL) and human apolipoprotein B (apoB) on low-density lipoprotein (LDL) subclasses were examined in rabbits, a species naturally deficient in HL activity. Methods and Results— In apoB-transgenic rabbit plasma, >80% of the protein was found in the 1.006- to 1.050-g/mL fraction. Gradient gel electrophoresis (GGE) of this fraction revealed two distinct species, designated large and small LDL. A denser fraction (d=1.050 to 1.063 g/mL) contained small LDL as well as another discrete LDL subspecies, designated very small LDL. Expression of HL resulted in reductions in protein concentrations in the 1.006- to 1.050-g/mL densi…
Myelin: Methods for Purification and Proteome Analysis
2019
Molecular characterization of myelin is a prerequisite for understanding the normal structure of the axon/myelin-unit in the healthy nervous system and abnormalities in myelin-related disorders. However, reliable molecular profiles necessitate very pure myelin membranes, in particular when considering the power of highly sensitive "omics"-data acquisition methods. Here, we recapitulate the history and recent applications of myelin purification. We then provide our laboratory protocols for the biochemical isolation of a highly pure myelin-enriched fraction from mouse brains and for its proteomic analysis. We also supply methodological modifications when investigating posttranslational modifi…
Assays of Proteasome-Dependent Cleavage Products
2005
The degradation of misfolded, aged, or no longer needed cytosolic proteins depends largely on the ubiquitin-proteasome system. Proteasomes degrade their substrates into fragments of 3-20 amino acids. Human 20S proteasomes can be purified from human erythrocytes by batch adsorption to DEAE-cellulose, ammonium sulfate precipitation, anion-exchange fast protein liquid chromatography (FPLC), and glycerol density gradient ultracentrifugation. 20S proteasomes purified by this method are suitable for the in vitro digestion of synthetic peptides as well as full-length proteins. The degradation products produced by proteasomes are separated by reversed-phase HPLC using an acetonitrile gradient. The …
Assembly and Separation of Semiconductor Quantum Dot Dimers and Trimers
2011
Repeated precipitation of colloidal semiconductor quantum dots (QD) from a good solvent by adding a poor solvent leads to an increasing number of QD oligomers after redispersion in the good solvent. By using density gradient ultracentrifugation we have been able to separate QD monomer, dimer, and trimer fractions from higher oligomers in such solutions. In the corresponding fractions QD dimers and trimers have been enriched up to 90% and 64%, respectively. Besides directly coupled oligomers, QD dimers and trimers were also assembled by linkage with a rigid terrylene diimide dye (TDI) and separated again by ultracentrifugation. High-resolution transmission electron micrographs show that the …
Low-density lipoproteins generated during an oral fat load in mild hypertriglyceridemic and healthy subjects are smaller, denser, and have an increas…
2005
Triglyceride-rich lipoproteins generated during the postprandial phase are atherogenic. Large very low-density lipoproteins (LDLs) or chylomicrons (CMs) are not as atherogenic as their remnants (Rem). Small and dense LDLs are associated with cardiovascular disease. Low-density lipoprotein size is partly under genetic control and is considered as a relatively stable LDL feature. In this article, we present data on retinyl palmitate kinetics correlated with the modification of LDL features in terms of size, density, and in vitro receptor binding affinity after an oral fat load. Six nondiabetic, hypertriglyceridemic (HTG) patients and 6 healthy controls were examined. Low-density lipoprotein s…
The production of 85 kDa N-terminal fragment of apolipoprotein B in mutant HepG2 cells generated by targeted modification of apoB gene occurs by ALLN…
2010
Abstract To study the mechanism of low levels of full length and truncated apoB in individuals heterozygous for apoB truncation, a non-sense mutation was introduced in one of the three alleles of apob gene of HepG2 cells by homologous recombination. Despite very low levels of apoB-82 (1–2%) in the media, a prominent N-terminal apoB protein of 85 kDa (apoB-15) was secreted that fractionated at d > 1.065 in density gradient ultracentrifugation. The mechanism of production of this short protein was studied by 35S-methionine pulse–chase experiment. Oleate prevented presecretory degradation of apoB-100 in the cell and resulted in increased secretion of newly synthesized apoB-100 with decreases i…
In vivo metabolism of LDL subfractions in patients with heterozygous FH on statin therapy
2004
LDL can be subfractionated into buoyant (1.020-1.029 g/ml(-1)), intermediate (1.030-1.040 g/ml(-1)), and dense (1.041-1.066 g/ml(-1)) LDLs. We studied the rebound of these LDL-subfractions after LDL apheresis in seven patients with heterozygous familial hypercholesterolemia (FH) regularly treated by apheresis (58 +/- 9 years, LDL-cholesterol = 342 +/- 87 mg/dl(-1), triglycerides = 109 +/- 39 mg/dl(-1)) and high-dose statins. Apolipoprotein B (apoB) concentrations were measured in LDL subfractions immediately after and on days 1, 2, 3, 5, and 7 after apheresis. Compartmental models were developed to test three hypotheses: 1) that dense LDLs are derived from the delipidation of buoyant and in…
Isolation of nuclei and downstream processing of cell-type-specific nuclei from micro-dissected mouse brain regions – techniques and caveats
2020
AbstractThe mammalian brain consists of several structurally and functionally distinct regions equipped with an equally complex cell-type system. Due to its relevance in uncovering disease mechanisms, the study of cell-type-specific molecular signatures of different brain regions has increased. The rapid evolution of newer and cheaper sequencing techniques has also boosted the interest in cell-type-specific epigenetic studies. In fact, the nucleus holds most of the cell’s epigenetic information and is quite resistant to tissue dissociation processes as compared to cells. As such, nuclei are continually preferred over cells for epigenetic studies. However, the isolation of nuclei from cells …