Purifying Viruses Using Sucrose Cushion v1

protocols.io ◽  
2015 ◽  
Author(s):  
Shannon Williamson
Keyword(s):  
Sucrose Cushion

Semi-purification procedures of prions from a prion-infected brain using sucrose has no influence on the nonenzymatic glycation of the disease-associated prion isoform

2016 ◽  
Vol 397 (2) ◽  
pp. 125-133 ◽  
Author(s):  
Yeong-Gon Choi ◽  
Jae-Il Kim ◽  
Eun-Kyoung Choi ◽  
Richard I. Carp ◽  
Yong-Sun Kim
Keyword(s):  
Colloidal Silica ◽  
Protein S ◽  
Core Region ◽  
Advanced Glycation ◽  
Nonenzymatic Glycation ◽  
Pellet Fraction ◽  
Sucrose Cushion ◽  
Advanced Glycation End Product ◽  
Modified Polymer

Abstract Previous studies have shown that the Nε-carboxymethyl group is linked to not only one or more N-terminal Lys residues but also to one or more Lys residues of the protease-resistant core region of the pathogenic prion isoform (PrPSc) in prion-infected brains. Using an anti-advanced glycation end product (AGE) antibody, we detected nonenzymatically glycated PrPSc (AGE-PrPSc) in prion-infected brains following concentration by a series of ultracentrifugation steps with a sucrose cushion. In the present study, the levels of in vitro nonenzymatic glycation of PrPSc using sucrose were investigated to determine whether sucrose cushion can artificially and nonenzymatically induce in vitro glycation during ultracentrifugation. The first insoluble pellet fraction following the first ultracentrifugation (PU1st) collected from 263K scrapie-infected brains was incubated with sucrose, glucose or colloidal silica coated with polyvinylpyrrolidone (percoll). None of the compounds in vitro resulted in AGE-PrPSc. Nonetheless, glucose and percoll produced AGEs in vitro from other proteins within PU1st of the infected brains. This reaction could lead to the AGE-modified polymer(s) of nonenzymatic glycation-prone protein(s). This study showed that PrPSc is not nonenzymatically glycated in vitro with sucrose, glucose or percoll and that AGE-modified PrPSc can be isolated and enriched from prion-infected brains.

scholarly journals A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection

2016 ◽  
Vol 36 (12) ◽  
pp. 1171-1177 ◽  
Author(s):  
Taís F. Cruz ◽  
Tatiana M. Kanashiro ◽  
Alessandra M.M.G. de Castro ◽  
Cintia M. Baldin ◽  
Leonardo J. Richtzenhain ◽  
...  
Keyword(s):  
Cell Culture ◽  
Natural Infection ◽  
Sandwich Elisa ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Antibody Detection ◽  
Porcine Circovirus ◽  
Sucrose Cushion ◽  
Double Antibody

ABSTRACT: Few studies have described enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against porcine circovirus type 2 (PCV2) based on antigens produced in cell culture. Furthermore, few articles have described viral purification techniques for members of the family Circoviridae. This occurs because circoviruses are difficult to isolate, noncytopathogenic, and produce low viral titres in cell culture. Thus, for overcoming these difficulties in the cultivation of PCV2, this study aimed to develop a double-antibody sandwich ELISA based on the cell culture antigen PCV2b for the quantification of anti-PCV2 antibodies. A 20% and 50% discontinuous sucrose cushion was used for viral purification, which enabled the separation of cell culture proteins in the 20% sucrose cushion and a greater viral concentration in the 50% sucrose cushion. Following isopycnic centrifugation, PCV2 was concentrated in the band with density values from 1.330 to 1.395g/cm3. Viral purification was assessed using SDS-PAGE, indirect ELISA and electron microscopy. The standardised ELISA revealed a strong linear correlation (r= 0.826, p<0.001) when compared with a commercial ELISA kit. The assay exhibited low variability (inter-assay coefficient of variation of 4.24% and intra-assay of 1.80%) and excellent analytical specificity conferred by the capture antibody produced in rabbit. Thus, this ELISA is a rapid, specific and convenient method for the detection of antibodies against PCV2 in studies of experimental and natural infection, and in monitoring the response to vaccination on commercial farms.

scholarly journals An improvised one-step sucrose cushion ultracentrifugation method for exosome isolation from culture supernatants of mesenchymal stem cells

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Suchi Gupta ◽  
Sonali Rawat ◽  
Vivek Arora ◽  
Sarat Kumar Kottarath ◽  
Amit Kumar Dinda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Sucrose Cushion ◽  
One Step ◽  
Culture Supernatants

Quantitative proteomics of human blood exosomes

2018 ◽  
Vol 64 (6) ◽  
pp. 496-504 ◽  
Author(s):  
N.A. Shushkova ◽  
N.E. Vavilov ◽  
S.E. Novikova ◽  
T.E. Farafonova ◽  
O.V. Tikhonova ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
Biological Fluids ◽  
Protein Composition ◽  
Membrane Vesicles ◽  
The Body ◽  
Parent Cell ◽  
Protein Biomarkers ◽  
Sucrose Cushion ◽  
Centrifugation Method ◽  
Almost All

Exosomes are extracellular membrane vesicles secreted by cells into biological fluids. The outer membrane of exosomes protects their content from degradation and contains markers of the parent cell. Almost all cells of the body produce exosomes, however, tumor cells secrete them more intensively. Due to fact that exosomes contain proteins of cells secreting them, these vesicles could be a valuable source for biomarkers discovery. Currently, a number of studies prove the participation of exosomes in carcinogenesis. However, there is a problem of isolating pure and characterized exosomes for further use in investigation of functions or identification of tumor protein biomarkers. In this work, we have performed experiments on exosomes isolation from human plasma by three methods: differential ultracentrifugation, ultracentrifugation in sucrose cushion, sedimentation of the exosomal fraction from serum by using a commercial kit. The protein composition of the obtained samples was determined by mass spectrometric methods of selected reactions monitoring (SRM) and shotgun proteomic analysis. The obtained exosomal samples were searched for the presence of exosomal markers (CD9, CD82, HSPA8, CD63). In the samples of exosomes isolated by ultracentrifugation with the sucrose cushion, the content of the above markers was determined as 32.85, 15.59, 6.07 fmol/mg of total protein, correspondently. It was shown that the centrifugation method with the sucrose cushion was optimal for the isolation of exosomes.

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