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Isolation of endoplasmic reticulum, mitochondria, and mitochondria-associated membrane fractions from transfected cells and from human cytomegalovirus-infected primary fibroblasts.
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- Additional Information
- Source:
Country of Publication: United States NLM ID: 101287856 Publication Model: Print Cited Medium: Internet ISSN: 1934-2616 (Electronic) Linking ISSN: 19342616 NLM ISO Abbreviation: Curr Protoc Cell Biol Subsets: MEDLINE
- Subject Terms:
- Abstract:
Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub-domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria-associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated. Because of its documented role in lipid and ceramide transfer from the ER to mitochondria, a method to purify MAM from infected cells was also developed. Two robust procedures were developed to efficiently isolate mitochondria, ER, and MAM fractions while providing the substantial protein yields from HCMV-infected primary fibroblasts and from transfected HeLa cells. Moreover, this unit includes a protocol that allows visualization of the mitochondria network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients.
((c) 2007 by John Wiley & Sons, Inc.)
- Accession Number:
0 (Immediate-Early Proteins)
0 (UL37 protein, Human herpesvirus 5)
0 (Viral Proteins)
- Publication Date:
Date Created: 20080130 Date Completed: 20080320 Latest Revision: 20161021
- Publication Date:
20221213
- Accession Number:
10.1002/0471143030.cb0327s37
- Accession Number:
18228515
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