Enrichment of Golgi membranes from HeLa cells by sucrose gradient ultracentrifugation

BIO-PROTOCOL ◽  
2013 ◽  
Vol 3 (18) ◽  
Author(s):  
Josse Galen ◽  
Julia Blume
Keyword(s):  
Hela Cells ◽  
Sucrose Gradient ◽  
Golgi Membranes ◽  
Sucrose Gradient Ultracentrifugation

Abstract 488: Sinoatrial Nodal Pacemaker Cells (SANC) Exhibit High Basal CA Activated Adenylyl Cyclase (AC) Activity and Express Multiple Distinctly Localized AC Types

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
David R Graham ◽  
Antoine Younes ◽  
Alexey Lyashkov ◽  
Anna Sheydina ◽  
Maria Volkova ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Sucrose Gradient ◽  
Soluble Fraction ◽  
Membrane Microdomains ◽  
Membrane Domains ◽  
Lipid Domains ◽  
Rt Pcr ◽  
Pacemaker Cells ◽  
Pacemaker Function ◽  
Sucrose Gradient Ultracentrifugation

In SANC constituitive AC generates high basal cAMP, inducing PKA-dependent phosphorylation that regulates Ca2+ cycling, that is essential for normal pacemaker function. Our goals were to identify, in rabbit SANC, the types of AC expressed, and their Ca2+ sensitivity and location. Radioimmunoassay (with total phosphodiasterase inhibition) showed a high Ca2+ activated basal AC activity. AC activity increased 5-fold from Ca2+ free (EGTA) to 1 uM free Ca2+. RT PCR (using specifically designed rabbit primers) showed that AC types II and V, and Ca2+ activated types, I and VIII, are expressed in SANC. The organization of these distinct AC types within calveolar or non-calveolar membrane microdomains was determined in pooled SANC isolated from 5 hearts, using triton x100, and sucrose gradient ultracentrifugation. Lipid domains segregated into caveolin containing and non-caveolin containing membrane microdomains, where AC activity was concentrated (fig , AC activity). Immunoblots demonstrated localization of different AC types between these two membrane domains, with AC I, II, V/VI localizing to caveolin containing lipid rafts, and AC VIII present in both caveolin and GM1 lipid domains, and also in the soluble fraction (fig ). In summary, multiple ACs, both Ca2+ activated and non-CA2+ activated types, are expressed in SANC, and these reside in distinct calveolar and non-calveolar lipid domains. We conclude that constituitive basal AC activity is, generated, in part, at least, by a Ca2+ activated AC. type.

scholarly journals Structural features of the first component of human complement, C1, as revealed by surface iodination

1982 ◽  
Vol 203 (1) ◽  
pp. 185-191 ◽  
Author(s):  
C L Villiers ◽  
S Chesne ◽  
M B Lacroix ◽  
G J Arlaud ◽  
M G Colomb
Keyword(s):  
Sucrose Gradient ◽  
Structural Features ◽  
Complement Component ◽  
Monomeric Form ◽  
Human Complement ◽  
Equal Distribution ◽  
A Chain ◽  
Sucrose Gradient Ultracentrifugation ◽  
Label Distribution ◽  
Human Complement Component

Lactoperoxidase-catalysed surface iodination and sucrose-gradient ultracentrifugation were used to investigate the structure of human complement component C1. 1. Proenzymic subcomponents C1r and C1s associated to form a trimeric C1r2-C1s complex (7.6 S) in the presence of EDTA, and a tetrameric Clr2-C1s2 complex (9.1 S) in the presence of Ca2+. Iodination of the 9.1 S complex led to a predominant labelling of C1r (70%) over C1s (30%), essentially located in the b-chain moiety of C1r and in the a-chain moiety of C1s. 2. Reconstruction of proenzymic soluble C1 (15.2 S) from C1q, C1r and C1s was partially inhibited when C1s labelled in its monomeric form was used and almost abolished when iodinated C1r was used. Reconstruction of fully activated C1 was not possible, whereas hybrid C1q-C1r2-C1s2 complex was obtained. 3. Iodination of proenzymic or activated C1 bound to IgG-ovalbumin aggregates led to an equal distribution of the radioactivity between C1q and C1r2-C1s2. With regard to C1q, the label distribution between the three chains was similar whether C1 was in its proenzymic or activated form. Label distribution in the C1r2-C1s2 moiety of C1 was the same as that obtained for isolated C1r2-C1s2, and this was also true for the corresponding activated components. However, two different labelling patterns were found, corresponding to the proenzyme and the activated states.

Apparent absence of symmetrical transcription in Tetrahymena mitochondria

1978 ◽  
Vol 56 (2) ◽  
pp. 107-110
Author(s):  
Paul G. Young
Keyword(s):  
Ionic Strength ◽  
Hela Cells ◽  
Sucrose Gradient ◽  
Pulse Length ◽  
Tetrahymena Pyriformis ◽  
Ribonuclease A ◽  
High Ionic Strength ◽  
Apparent Absence ◽  
Double Stranded Rna ◽  
Rapid Processing

Transcription in Tetrahymena pyriformis mitochondria has been examined for whether or not symmetrical transcription occurs. A ribonuclease A digestion in high-ionic strength was used as a test for the presence of double-stranded RNA (dsRNA) either before or after self-annealing- This test was applied to RNA prepared using pulse lengths of from 1 to 30 min. If symmetrical transcription occurred, then the amount of dsRNA should have varied inversely with pulse length. There was no evidence for the presence of dsRNA other than a ribonuclease-resistant core. A search was also made among the largest transcripts present (> 26S) since this region of a sucrose gradient is enriched in symmetrical transcripts in HeLa cells. Again none were found. In order to allow for possible rapid processing as the reason for the failure to find dsRNA, total cellular RNA was extracted as quickly as possible, and the ribonuclease digest analyzed by Sephadex chromatography. No large pieces of dsRNA were found.

Fractionation and antigenic characterization of organelles ofEimeria tenellasporozoites

Parasitology ◽  
1992 ◽  
Vol 104 (1) ◽  
pp. 1-9 ◽  
Author(s):  
U. Kawazoe ◽  
F. M. Tomley ◽  
J. A. Frazier
Keyword(s):  
Molecular Weight ◽  
Western Blotting ◽  
Sucrose Gradient ◽  
Eimeria Tenella ◽  
2Nd Generation ◽  
Antigenic Characterization ◽  
Electron Microscopical ◽  
Sucrose Gradient Ultracentrifugation ◽  
Antigenic Reactivity

SUMMARYSporozoites ofEimeria tenellawere disrupted by sonication and subcellular fractions were separated by sucrose gradient ultracentrifugation. Fractions from gradients were characterized by electron microscopical appearance and their polypeptide and antigenic profiles determined by PAGE and Western blotting with antisera to sporozoites and 1st- and 2nd-generation merozoites. Fractions containing micronemes, rhoptries or membranes showed markedly different polypeptide content and antigenic reactivity. Microneme epitopes were strongly conserved between sporozoites and 2nd-generation merozoites whereas the majority of rhoptry epitopes and many membrane epitopes were sporozoite specific. The only polypeptide of sporozoites which was strongly recognized by antisera raised to 1st generation merozoites was a microneme antigen of molecular weight approximately 100 kDa.

scholarly journals Comparison of methods for purification of bacteriophage lysates of gram-negative bacteria for personalized therapy

2021 ◽  
Author(s):  
RB Gorodnichev ◽  
MA Kornienko ◽  
NS Kuptsov ◽  
AD Efimov ◽  
VI Bogdan ◽  
...  
Keyword(s):  
Density Gradient ◽  
Sucrose Gradient ◽  
Sucrose Density Gradient ◽  
Density Gradient Ultracentrifugation ◽  
Phage Lysate ◽  
Purification Method ◽  
Cscl Density Gradient ◽  
Cell Components ◽  
Purification Methods ◽  
Sucrose Gradient Ultracentrifugation

Phage therapy is a promising method of treating antibiotic-resistant infections. To obtain a safe therapeutic formulation, bacterial cell components, including endotoxins, must be removed from the phage lysate. This study was aimed at comparing the efficacy of purification methods for phage lysates intended for therapeutic use. Phages vB_KpnM_Seu621 (Myoviridae) and vB_KpnP_Dlv622 (Autographiviridae) were grown using the KP9068 strain of Klebsiella pneumoniae as a host. The obtained lysates were purified using phage precipitation with polyethylene glycol, CsCl density gradient ultracentrifugation, sucrose density gradient ultracentrifugation, precipitation with 100 kDa centrifugal filter units, and phage concentration on 0.22 µm cellulose filters in the presence of MgSO4. Endotoxin concentrations were determined by LAL testing. The obtained lysates contained 1.25 × 1012 ± 7.46 × 1010 and 2.25 × 1012 ± 1.34 × 1011 PFU/ml of vB_KpnM_Seu621 and vB_KpnP_Dlv622, respectively, and had endotoxin concentrations of 3,806,056 ± 429,410 and 189,456 ± 12,406 EU/ml, respectively. CsCl gradient ultracentrifugation was found to be the optimal conventional purification method in terms of reducing endotoxin concentrations and maintaining phage titers (303 ± 20 — 313 ± 35 EU/ml, 1.5–2.75 × 1012 ± 1.71 × 1011 PFU/ml). Sucrose gradient ultracentrifugation and filtration in the presence of MgSO4 were found to be the optimal non-traditional purification methods. A method for phage lysate purification should be selected for each phage preparation individually. Sucrose gradient ultracentrifugation and filtration in the presence of MgSO4 hold promise as purification methods that can produce phage preparations suitable for intravenous administration.

Isolation and Analysis of Bacterial Ribosomes Through Sucrose Gradient Ultracentrifugation

2019 ◽  
pp. 299-310
Author(s):  
Ricardo F. dos Santos ◽  
Cátia Bárria ◽  
Cecília M. Arraiano ◽  
José M. Andrade
Keyword(s):  
Sucrose Gradient ◽  
Sucrose Gradient Ultracentrifugation
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