Sucrose density gradient centrifugation Dotaz Zobrazit nápovědu
- MeSH
- buněčné linie MeSH
- centrifugace - gradient hustoty MeSH
- druhová specificita MeSH
- elektroforéza MeSH
- Herpesviridae izolace a purifikace patogenita růst a vývoj MeSH
- kultivace virů MeSH
- kultivační média MeSH
- kuřecí embryo MeSH
- ledviny MeSH
- metody MeSH
- prasata MeSH
- pseudovzteklina MeSH
- sacharosa MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- kuřecí embryo MeSH
- skot MeSH
- zvířata MeSH
Velocity separation of translation complexes in linear sucrose gradients is the ultimate method for both analysis of the overall fitness of protein synthesis as well as for detailed investigation of physiological roles played by individual factors of the translational machinery. Polysome profile analysis is a frequently performed task in translational control research that not only enables direct monitoring of the efficiency of translation but can easily be extended with a wide range of downstream applications such as Northern and Western blotting, genome-wide microarray analysis or qRT-PCR. This chapter provides a basic overview of the polysome profile analysis technique and the RNA isolation procedure from sucrose gradients. We also discuss possible experimental pitfalls of data normalization, describe main alternatives of the basic protocol and outline a novel application of denaturing RNA electrophoresis in several steps of polysome profile analysis.
- MeSH
- centrifugace - gradient hustoty metody MeSH
- elektroforéza metody MeSH
- interpretace statistických dat MeSH
- kvasinky MeSH
- polyribozomy chemie MeSH
- proteosyntéza genetika MeSH
- regulace genové exprese genetika MeSH
- RNA izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- barvení a značení MeSH
- bezbuněčný systém MeSH
- buněčné linie MeSH
- centrifugace - gradient hustoty MeSH
- centrifugace zonální MeSH
- elektronová mikroskopie MeSH
- fibroblasty MeSH
- kur domácí MeSH
- kuřecí embryo MeSH
- mitochondrie mikrobiologie MeSH
- nádorová transformace buněk MeSH
- ptačí sarkom mikrobiologie MeSH
- replikace viru MeSH
- sacharosa MeSH
- spektrofotometrie MeSH
- viry ptačího sarkomu izolace a purifikace patogenita růst a vývoj MeSH
- zvířata MeSH
- Check Tag
- kuřecí embryo MeSH
- zvířata MeSH
More than one 80S monosome can translate an mRNA molecule at a time producing polysomes. The most widely used method to separate 40S and 60S ribosomal subunits from 80S monosomes and polysomes is a high-velocity centrifugation of whole cell extracts in linear sucrose gradients. This polysome profile analysis technique has been routinely used to monitor translational fitness of cells under a variety of physiological conditions, to investigate functions of initiation factors involved in translation, to reveal defects in ribosome biogenesis, to determine roles of 5' UTR structures on mRNA translatability, and more recently for examination of miRNA-mediated translational repression (see an application of this protocol on Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae).
- MeSH
- centrifugace - gradient hustoty MeSH
- centrifugace MeSH
- chemická precipitace MeSH
- chlazení MeSH
- chromatografie MeSH
- dialýza MeSH
- fosfáty MeSH
- glykoly MeSH
- halogenované uhlovodíky MeSH
- jedlé rostliny MeSH
- koncentrace vodíkových iontů MeSH
- merkaptoethanol MeSH
- metody MeSH
- nemoci rostlin MeSH
- pufry MeSH
- rostlinné extrakty MeSH
- rostlinné viry izolace a purifikace patogenita MeSH
- sacharasa MeSH
- MeSH
- centrifugace - gradient hustoty MeSH
- centrifugace MeSH
- chemická precipitace MeSH
- chlazení MeSH
- chromatografie MeSH
- dialýza MeSH
- fosfáty MeSH
- glykoly MeSH
- halogenované uhlovodíky MeSH
- jedlé rostliny MeSH
- koncentrace vodíkových iontů MeSH
- merkaptoethanol MeSH
- metody MeSH
- nemoci rostlin MeSH
- pufry MeSH
- rostlinné extrakty MeSH
- rostlinné viry izolace a purifikace patogenita MeSH
- sacharosa MeSH
We present a simple method for enrichment of lysosomal membranes from HEK293 and HeLa cell lines taking advantage of selective disruption of lysosomes by methionine methyl ester. Organelle concentrate from postnuclear supernatant was treated with 20 mmol/l methionine methyl ester for 45 min to lyse the lysosomes. Subsequently, lysosomal membranes were resolved on a step sucrose gradient. An enriched lysosomal membrane fraction was collected from the 20%/35% sucrose interface. The washed lysosomal membrane fraction was enriched 30 times relative to the homogenate and gave the yield of more than 8%. These results are comparable to lysosomal membranes isolated by magnetic chromatography from cultured cells (Diettrich et al., 1998). The procedure effectively eliminated mitochondrial contamination and minimized contamination from other cell compartments. The enriched fractions retained the ability to acidify membrane vesicles through the activity of lysosomal vacuolar ATPase. The method avoids non-physiological overloading of cells with superparamagnetic particles and appears to be quite robust among the tested cell lines. We expect it may be of more general use, adaptable to other cell lines and tissues.
- MeSH
- adenosintrifosfát farmakologie MeSH
- centrifugace - gradient hustoty MeSH
- frakcionace buněk metody MeSH
- glukosylceramidasa metabolismus MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- intracelulární membrány účinky léků metabolismus MeSH
- kyseliny metabolismus MeSH
- lidé MeSH
- lyzozomy účinky léků metabolismus MeSH
- subcelulární frakce účinky léků metabolismus MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
