Immunoisolation Using Magnetic Solid Supports: Subcellular Fractionation for Cell-Free Functional Studies

1991 ◽  
pp. 171-198
Author(s):  
KATHRYN E. HOWELL ◽  
RUTH SCHMID ◽  
JOHN UGELSTAD ◽  
JEAN GRUENBERG
Keyword(s):  
Subcellular Fractionation ◽  
Solid Supports ◽  
Functional Studies ◽  
Magnetic Solid

scholarly journals Simple and Efficient Protocol for Subcellular Fractionation of Normal and Apoptotic Cells

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 852
Author(s):  
Viacheslav V. Senichkin ◽  
Evgeniia A. Prokhorova ◽  
Boris Zhivotovsky ◽  
Gelina S. Kopeina
Keyword(s):  
Subcellular Fractionation ◽  
Nuclear Fraction ◽  
Apoptotic Cells ◽  
Apoptotic Bodies ◽  
Functional Studies ◽  
Ionic Detergents ◽  
Protein Functions ◽  
Indispensable Tool ◽  
Cell Fragments

Subcellular fractionation approaches remain an indispensable tool among a large number of biochemical methods to facilitate the study of specific intracellular events and characterization of protein functions. During apoptosis, the best-known form of programmed cell death, numerous proteins are translocated into and from the nucleus. Therefore, suitable biochemical techniques for the subcellular fractionation of apoptotic cells are required. However, apoptotic bodies and cell fragments might contaminate the fractions upon using the standard protocols. Here, we compared different nucleus/cytoplasm fractionation methods and selected the best-suited approach for the separation of nuclear and cytoplasmic contents. The described methodology is based on stepwise lysis of cells and washing of the resulting nuclei using non-ionic detergents, such as NP-40. Next, we validated this approach for fractionation of cells treated with various apoptotic stimuli. Finally, we demonstrated that nuclear fraction could be further subdivided into nucleosolic and insoluble subfractions, which is crucial for the isolation and functional studies of various proteins. Altogether, we developed a method for simple and efficient nucleus/cytoplasm fractionation of both normal and apoptotic cells.

scholarly journals SIRPβ1 Is Expressed as a Disulfide-linked Homodimer in Leukocytes and Positively Regulates Neutrophil Transepithelial Migration

2005 ◽  
Vol 280 (43) ◽  
pp. 36132-36140 ◽  
Author(s):  
Yuan Liu ◽  
Ileana Soto ◽  
Qiao Tong ◽  
Alex Chin ◽  
Hans-Jörg Bühring ◽  
...  
Keyword(s):  
Cell Surface ◽  
Adaptor Proteins ◽  
Subcellular Fractionation ◽  
Fine Tuning ◽  
Human Neutrophils ◽  
Functional Studies ◽  
Inflammatory Stimuli ◽  
Signaling Receptors ◽  
Cell Surface Signaling ◽  
And Function

Signal regulatory proteins (SIRPs) comprise a family of cell surface signaling receptors differentially expressed in leukocytes and the central nervous system. Although the extracellular domains of SIRPs are highly similar, classical motifs in the cytoplasmic or transmembrane domains distinguish them as either activating (β) or inhibitory (α) isoforms. We reported previously that human neutrophils (polymorphonuclear leukocytes (PMN)) express multiple SIRP isoforms and that SIRPα binding to its ligand CD47 regulates PMN transmigration. Here we further characterized the expression of PMN SIRPs, and we reported that the major SIRPα and SIRPβ isoforms expressed in PMN include Bit/PTPNS-1 and SIRPβ1, respectively. Furthermore, although SIRPα (Bit/PTPNS-1) is expressed as a monomer, we showed that SIRPβ1 is expressed on the cell surface as a disulfide-linked homodimer with bond formation mediated by Cys-320 in the membrane-proximal Ig loop. Subcellular fractionation studies revealed a major pool of SIRPβ1 within the plasma membrane fractions of PMN. In contrast, the majority of SIRPα (Bit/PTPNS-1) is present in fractions enriched in secondary granules and is translocated to the cell surface after chemoattractant (formylmethionylleucylphenylalanine) stimulation. Functional studies revealed that antibody-mediated ligation of SIRPβ1 enhanced formylmethionylleucylphenylalanine-driven PMN transepithelial migration. Co-immunoprecipitation experiments to identify associated adaptor proteins revealed a 10–12-kDa protein associated with SIRPβ1 that was tyrosine-phosphorylated after PMN stimulation and is not DAP10/12 or Fc receptor γ chain. These results provide new insights into the structure and function of SIRPs in leukocytes and their potential role(s) in fine-tuning responses to inflammatory stimuli.

scholarly journals Isolation of 3.5-kb Fragments on Magnetic Solid Supports

BioTechniques ◽  
1996 ◽  
Vol 20 (2) ◽  
pp. 198-200 ◽  
Author(s):  
Steve K. Kotsopoulos ◽  
Anthony P. Shuber
Keyword(s):  
Solid Supports ◽  
Magnetic Solid

scholarly journals The Diabetes Autoantigen ICA69 and Its Caenorhabditis elegans Homologue, ric-19, Are Conserved Regulators of Neuroendocrine Secretion

2000 ◽  
Vol 11 (10) ◽  
pp. 3277-3288 ◽  
Author(s):  
Marc Pilon ◽  
Xiao-Rong Peng ◽  
Andrew M. Spence ◽  
Ronald H.A. Plasterk ◽  
Hans-Michael Dosch
Keyword(s):  
Caenorhabditis Elegans ◽  
Model Organism ◽  
Secretory Activity ◽  
Subcellular Fractionation ◽  
Secretory Cells ◽  
Secretory Vesicles ◽  
Expression Levels ◽  
C Elegans ◽  
Functional Studies ◽  
Neurotransmitter Secretion

ICA69 is a diabetes autoantigen with no homologue of known function. Given that most diabetes autoantigens are associated with neuroendocrine secretory vesicles, we sought to determine if this is also the case for ICA69 and whether this protein participates in the process of neuroendocrine secretion. Western blot analysis of ICA69 tissue distribution in the mouse revealed a correlation between expression levels and secretory activity, with the highest expression levels in brain, pancreas, and stomach mucosa. Subcellular fractionation of mouse brain revealed that although most of the ICA69 pool is cytosolic and soluble, a subpopulation is membrane-bound and coenriched with synaptic vesicles. We used immunostaining in the HIT insulin-secreting β-cell line to show that ICA69 localizes in a punctate manner distinct from the insulin granules, suggesting an association with the synaptic-like microvesicles found in these cells. To pursue functional studies on ICA69, we chose to use the model organism Caenorhabditis elegans, for which a homologue of ICA69 exists. We show that the promoter of the C. elegans ICA69 homologue is specifically expressed in all neurons and specialized secretory cells. A deletion mutant was isolated and found to exhibit resistance to the drug aldicarb (an inhibitor of acetylcholinesterase), suggesting defective neurotransmitter secretion in the mutant. On the basis of the aldicarb resistance phenotype, we named the gene ric-19 (resistance to inhibitors of cholinesterase-19). The resistance to aldicarb was rescued by introducing a ric-19 transgene into theric-19 mutant background. This is the first study aimed at dissecting ICA69 function, and our results are consistent with the interpretation that ICA69/RIC-19 is an evolutionarily conserved cytosolic protein participating in the process of neuroendocrine secretion via association with certain secretory vesicles.

Immunoisolation of Organelles Using Magnetic Solid Supports

Cell Biology ◽  
2006 ◽  
pp. 27-31 ◽  
Author(s):  
R FLUKIGERGAGESCU ◽  
J GRUENBERG
Keyword(s):  
Solid Supports ◽  
Magnetic Solid

The low-resolution 3D-structure of porin, a channel-forming protein in E. coliouter membranes

1983 ◽  
Vol 41 ◽  
pp. 440-441
Author(s):  
A. Engel ◽  
D.L. Dorset ◽  
A. Massalski ◽  
J.P. Rosenbusch
Keyword(s):  
Crystal Packing ◽  
3D Structure ◽  
Gram Negative Bacteria ◽  
Protein Ratio ◽  
Crystal Forms ◽  
Large Molecules ◽  
Functional Studies ◽  
Voltage Dependent ◽  
Planar Membranes ◽  
Hexagonal Arrays

Porins represent a group of channel forming proteins that facilitate diffusion of small solutes across the outer membrane of Gram-negative bacteria, while excluding large molecules (>650 Da). Planar membranes reconstituted from purified matrix porin (OmpF protein) trimers and phospholipids have allowed quantitative functional studies of the voltage-dependent channels and revealed concerted activation of triplets. Under the same reconstitution conditions but using high protein concentrations porin aggregated to 2D lattices suitable for electron microscopy and image processing. Depending on the lipid-to- protein ratio three different crystal packing arrangements were observed: a large (a = 93 Å) and a small (a = 79 Å) hexagonal and a rectangular (a = 79 Å b = 139 Å) form with p3 symmetry for the hexagonal arrays. In all crystal forms distinct stain filled triplet indentations could be seen and were found to be morphologically identical within a resolution of (22 Å). It is tempting to correlate stain triplets with triple channels, but the proof of this hypothesis requires an analysis of the structure in 3 dimensions.

Ionic homeostasis and subcellular element compartmentation

1990 ◽  
Vol 48 (2) ◽  
pp. 150-151
Author(s):  
Ann LeFurgey ◽  
Peter Ingram ◽  
J.J. Blum ◽  
M.C. Carney ◽  
L.A. Hawkey ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Ionic Homeostasis ◽  
X Ray ◽  
Functional Studies ◽  
Cell Compartments ◽  
X Ray Imaging ◽  
Resolution Electron ◽  
Multiple Cell ◽  
Role Of Zn

Subcellular compartments commonly identified and analyzed by high resolution electron probe x-ray microanalysis (EPXMA) include mitochondria, cytoplasm and endoplasmic or sarcoplasmic reticulum. These organelles and cell regions are of primary importance in regulation of cell ionic homeostasis. Correlative structural-functional studies, based on the static probe method of EPXMA combined with biochemical and electrophysiological techniques, have focused on the role of these organelles, for example, in maintaining cell calcium homeostasis or in control of excitation-contraction coupling. New methods of real time quantitative x-ray imaging permit simultaneous examination of multiple cell compartments, especially those areas for which both membrane transport properties and element content are less well defined, e.g. nuclei including euchromatin and heterochromatin, lysosomes, mucous granules, storage vacuoles, microvilli. Investigations currently in progress have examined the role of Zn-containing polyphosphate vacuoles in the metabolism of Leishmania major, the distribution of Na, K, S and other elements during anoxia in kidney cell nuclel and lysosomes; the content and distribution of S and Ca in mucous granules of cystic fibrosis (CF) nasal epithelia; the uptake of cationic probes by mltochondria in cultured heart ceils; and the junctional sarcoplasmic retlculum (JSR) in frog skeletal muscle.

Phytochemical and functional studies on the roots of Armoracia rusticana

2017 ◽  
Author(s):  
E Jimenez Negro ◽  
J Sendker ◽  
B Scharf ◽  
M Kleinwächter ◽  
B Lipowicz ◽  
...  
Keyword(s):  
Armoracia Rusticana ◽  
Functional Studies
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