Abstract:
:Dynamic interplay between intracellular organelles requires a particular functional apposition of membrane structures. The organelles involved come into close contact, but do not fuse, thereby giving rise to notable microdomains; these microdomains allow rapid communication between the organelles. Plasma membrane-associated membranes (PAMs), which are microdomains of the plasma membrane (PM) interacting with the endoplasmic reticulum (ER) and mitochondria, are dynamic structures that mediate transport of proteins, lipids, ions and metabolites. These structures have gained much interest lately owing to their roles in many crucial cellular processes. Here we provide an optimized protocol for the isolation of PAM, PM and ER fractions from rat liver that is based on a series of differential centrifugations, followed by the fractionation of crude PM on a discontinuous sucrose gradient. The procedure requires ∼8-10 h, and it can be easily modified and adapted to other tissues and cell types.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Suski JM,Lebiedzinska M,Wojtala A,Duszynski J,Giorgi C,Pinton P,Wieckowski MRdoi
10.1038/nprot.2014.016subject
Has Abstractpub_date
2014-02-01 00:00:00pages
312-22issue
2eissn
1754-2189issn
1750-2799pii
nprot.2014.016journal_volume
9pub_type
杂志文章相关文献
Nature Protocols文献大全abstract::This protocol describes the synthesis of oligo(poly(ethylene glycol) fumarate) (OPF; 1-35 kDa; a polymer useful for tissue engineering applications) by a one-pot reaction of poly(ethylene glycol) (PEG) and fumaryl chloride. The procedure involves three parts: dichloromethane and PEG are first dried; the reaction step ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2012.055
更新日期:2012-05-31 00:00:00
abstract::Several cell death assays have been developed based on a single biochemical parameter such as caspase activation or plasma membrane permeabilization. Our fluorescent apoptosis/necrosis (FAN) assay directly measures cell death and distinguishes between caspase-dependent apoptosis and caspase-independent necrosis of cel...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2016.085
更新日期:2016-08-01 00:00:00
abstract::Embryonic stem cells (ESCs) constitute a tool of great potential in neurobiology, enabling the directed differentiation of specific neural cell types. We have shown recently that neurons of the cerebral cortex can be generated from mouse ESCs cultured in a chemically defined medium that contains no morphogen, but in t...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2009.157
更新日期:2009-01-01 00:00:00
abstract::This protocol presents the peptide incorporation of environment-sensitive fluorophores derived from the dimethylaminophthalimide family. The procedure utilizes anhydride precursors of 4-dimethylaminophthalimide (4-DMAP) or 6-dimethylaminonaphthalimide (6-DMN), whose syntheses are described in a related protocol from t...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.442
更新日期:2007-01-01 00:00:00
abstract::In the past decade, Drosophila has emerged as an ideal model organism for studying the genetic components of sleep as well as its regulation and functions. In fruit flies, sleep can be conveniently estimated by measuring the locomotor activity of the flies using techniques and instruments adapted from the field of cir...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2012.041
更新日期:2012-04-26 00:00:00
abstract::Mass spectrometry (MS)-based proteomics has become the preferred tool for the analysis of protein phosphorylation. To be successful at such an endeavor, there is a requirement for an efficient enrichment of phosphopeptides. This is necessary because of the substoichiometric nature of phosphorylation at a given site an...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.010
更新日期:2013-03-01 00:00:00
abstract::Mouse embryonic stem cells (mESCs) are key tools for genetic engineering, development of stem cell-based therapies and basic research on pluripotency and early lineage commitment. However, successful derivation of germline-competent embryonic stem cell lines has, until recently, been limited to a small number of inbre...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2014.030
更新日期:2014-03-01 00:00:00
abstract::Many techniques have been developed for studying inducible gene expression, but all of them are multicomponent systems consisting of cis-acting elements at the DNA or RNA level, trans-acting regulator proteins and/or small molecules as inducers. RNA thermometers are the only known single-component regulators of gene e...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2009.112
更新日期:2009-01-01 00:00:00
abstract::Protein ubiquitination is a versatile protein modification that regulates virtually all cellular processes. This versatility originates from polyubiquitin chains, which can be linked in eight distinct ways. The combinatorial complexity of eight linkage types in homotypic (one chain type per polymer) and heterotypic (m...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2015.018
更新日期:2015-02-01 00:00:00
abstract::As proteins within cells are spatially organized according to their role, knowledge about protein localization gives insight into protein function. Here, we describe the LOPIT technique (localization of organelle proteins by isotope tagging) developed for the simultaneous and confident determination of the steady-stat...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.254
更新日期:2006-01-01 00:00:00
abstract::N-Succinimidyl 3-(di-tert-butyl[(18)F]fluorosilyl)benzoate ([(18)F]SiFB) is a highly reactive prosthetic group for radiolabeling of proteins for use in positron emission tomography (PET). It is similar to N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB), the 'gold-standard' prosthetic group for protein (18)F-labeli...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2012.110
更新日期:2012-11-01 00:00:00
abstract::Development of non-invasive and accurate methods to track cell fate after delivery will greatly expedite transition of embryonic stem (ES) cell therapy to the clinic. In this protocol, we describe the in vivo monitoring of stem cell survival, proliferation and migration using reporter genes. We established stable ES c...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2009.100
更新日期:2009-01-01 00:00:00
abstract::Here, we describe a simple micromolding method to construct three-dimensional arrays of organotypic epithelial tissue structures that approximate in vivo histology. An elastomeric stamp containing an array of posts of defined geometry and spacing is used to mold microscale cavities into the surface of type I collagen ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2008.35
更新日期:2008-01-01 00:00:00
abstract::The dynamics of transcription can be studied genome wide by high-throughput sequencing of nascent and newly synthesized RNA. 4-thiouridine (4SU) labeling in vivo enables the specific capture of such new transcripts, with 4SU residues being tagged by biotin linkers and captured using streptavidin beads before library p...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-019-0262-3
更新日期:2020-02-01 00:00:00
abstract::In this protocol, we describe cryoimmunolabeling methods for the subcellular localization of proteins and certain lipids. The methods start with chemical fixation of cells and tissue in formaldehyde (FA) and/or glutaraldehyde (GA), sometimes supplemented with acrolein. Cell and tissue blocks are then immersed in 2.3 M...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.365
更新日期:2007-01-01 00:00:00
abstract::Characterization of the cellular participants in tissue immune responses is crucial to understanding infection, cancer, autoimmunity, allergy, graft rejection and other immunological processes. Previous reports indicate that leukocytes in lung vasculature fail to be completely removed by perfusion. Several studies sug...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2014.005
更新日期:2014-01-01 00:00:00
abstract::Glycoproteins are involved in diverse biological processes ranging from extracellular contact and recognition to intracellular signaling. Crystal structures of glycoproteins would yield tremendous insight into these processes. But glycoprotein structural analysis has been hindered by difficulties in expressing milligr...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2009.29
更新日期:2009-01-01 00:00:00
abstract::High-throughput sequencing of the variable domains of immune receptors (antibodies and T cell receptors (TCRs)) is of key importance in the understanding of adaptive immune responses in health and disease. However, the sequencing of both immune receptor chains (VH+VL or TCRβ/δ+TCRα/γ) at the single-cell level for typi...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2016.024
更新日期:2016-03-01 00:00:00
abstract::The colorimetric bio-barcode assay is a red-to-blue color change-based protein detection method with ultrahigh sensitivity. This assay is based on both the bio-barcode amplification method that allows for detecting miniscule amount of targets with attomolar sensitivity and gold nanoparticle-based colorimetric DNA dete...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.201
更新日期:2007-01-01 00:00:00
abstract::This protocol describes a method for directing the expression of genes of interest into postmitotic neocortical neurons in vivo. Microinjection of a DNA plasmid-amphiphilic molecule mix into the neocortex followed by delivery of an ad hoc electric pulse protocol during the first few days of life in mice allows rapid, ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2015.001
更新日期:2015-01-01 00:00:00
abstract::Pseudo-afferent cervical lymph-duct cannulation in a sheep model allows large amounts of lymph cells to be collected under physiological conditions, carrying immune signaling information from the head tissues, including oro-nasal mucosae. Importantly, large quantities of dendritic cells (DCs) of several subtypes are o...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.147
更新日期:2006-01-01 00:00:00
abstract::Here we present a protocol for the extraction of RNA from Dictyostelium discoideum. Dictyostelium is a social amoeba that undergoes a basic developmental program, and therefore analysis of RNA levels over a time course is a commonly used technique. This procedure is similar to other guanidine thiocyanate-based methods...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.191
更新日期:2007-01-01 00:00:00
abstract::Positron emission tomography (PET) is a sensitive and noninvasive imaging method that is widely used to explore molecular events in living subjects. PET can precisely and quantitatively evaluate cellular apoptosis, which has a crucial role in various physiological and pathological processes. In this protocol, we descr...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2015.048
更新日期:2015-05-01 00:00:00
abstract::The use of anisotropic NMR data, such as residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs), has emerged as a powerful technique for structural characterization of organic small molecules. RDCs typically report the relative orientations of different 1H-13C bonds; RCSAs report the relati...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-018-0091-9
更新日期:2019-01-01 00:00:00
abstract::The terminal monosaccharide of cell surface glycoconjugates is typically a sialic acid (SA), and aberrant sialylation is involved in several diseases. Several methodological approaches in sample preparation and subsequent analysis using mass spectrometry (MS) have enabled the identification of glycosylation sites and ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2010.167
更新日期:2010-12-01 00:00:00
abstract::An important area of proteomics involves the need for quantification, whether relative or absolute. Many methods now exist for relative quantification, but to support biomarker proteomics and systems biology, absolute quantification rather than relative quantification is required. Absolute quantification usually invol...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.129
更新日期:2006-01-01 00:00:00
abstract::Cognitive computing is revolutionizing the way big data are processed and integrated, with artificial intelligence (AI) natural language processing (NLP) platforms helping researchers to efficiently search and digest the vast scientific literature. Most available platforms have been developed for biomedical researcher...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-020-00455-4
更新日期:2021-01-22 00:00:00
abstract::This protocol describes a method for the extraction of DNA from elephant ivory. These techniques are being used to assign geographic origin to poached ivory by comparing the ivory genotype to a geographic-based gene frequency map, developed separately. The method has three components: ivory pulverization, decalcificat...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.318
更新日期:2007-01-01 00:00:00
abstract::The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody-secreting plasmablasts can be enriched in vivo, in a severe combined immunode...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2014.104
更新日期:2014-07-01 00:00:00
abstract::The cytokinesis-block micronucleus cytome assay is a comprehensive system for measuring DNA damage, cytostasis and cytotoxicity. DNA damage events are scored specifically in once-divided binucleated (BN) cells and include (a) micronuclei (MNi), a biomarker of chromosome breakage and/or whole chromosome loss, (b) nucle...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.77
更新日期:2007-01-01 00:00:00