Isolation Of Endoplasmic Reticulum Research Articles

Isolation of Endoplasmic Reticulum and Its Membrane.

The association of ribosomes with the rough endoplasmic reticulum (ER) is dependent on Mg2+. The ribosomes can be stripped from the ER by removal of Mg2+ from the medium, resulting in a reduction in the ER membrane density and a diagnostic shift in migration when ER vesicles are analyzed by equilibrium density gradient centrifugation. Here, I describe the isolation of microsomes from Arabidopsis, followed by the use of the density shift approach in conjunction with equilibrium density gradient centrifugation as a means to diagnose whether a protein is associated with the ER. The same approach can also be used as a means to enrich for ER membranes.

Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts.

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 substantial protein yields from HCMV-infected primary fibroblasts and from transfected HeLa cells. Furthermore, this unit includes protocols for isolation of detergent resistant membranes from subcellular fractions as well as techniques that allow visualization of the mitochondrial network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients.

Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria‐Associated Membrane Fractions from Transfected Cells and from Human Cytomegalovirus‐Infected Primary Fibroblasts

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.

The isolation of endoplasmic reticulum from barley aleurone layers

Techniques for the isolation and purification of endoplasmic reticulum (ER) from aleurone layers of barley (Hordeum vulgare L.) were assessed. Neither differential centrifugation nor density gradient centrifugation of a homogenate separate the ER or other organelles of this tissue from the lipidcontaining spherosomes. Isopycnic sucrose gradient centrifugation of organelles first purified by molecular sieve chromatography on Sepharose 4B, however, results in separation of the organelles based on their differing buoyant densities. Manipulation of the magnesium concentration of the isolation media and density-gradient solutions affords isolation of ER at a density of 1.13-1.14 g cc(-1) and 1.17-1.18 g cc(-1). Electron microscopy shows that the membranes sedimenting at 1.13-1.14 g cc(-1) are devoid of ribosomes and are characteristic of smooth ER, while those sedimenting at 1.17-1.18 g cc(-1) are studded with ribosomes and have the features of rough ER. Endoplasmic reticulum isolated by isopycnic density gradient centrifugation can be further purified by rate-zonal centrifugation.

Sequestration of protein by the fat body of an insect.

THE fat body in an insect commonly contains a single cell type which is functionally diverse. At various times it stores and mobilizes fat, protein and glycogen which it may have synthesized or sequestered. In general the fat body becomes filled with reserves as metamorphosis approaches1. Larvae of Aedes fed on casein build up reserves of protein granules in the fat body2, and in Drosophila larvae, protein granules are induced to form in the middle of the third stadium by a hormone from the ring gland3. In Calpodes larvae the protein granules are probably translocated endocuticle. They arise suddenly within the Golgi vesicles 30–35 h before pupation, at a time when the thick larval endocuticle is being resorbed by the moulting fluid. At about 10 h before pupation other granules containing both protein and ribosomes arise from the isolation of endoplasmic reticulum in cytolysomes4. Although the formation and occurrence of protein granules in the fat body are well documented, there is little information on the origin of the protein.