Nuclear isolation buffer (NIB; 2X)

2008 ◽  
Vol 2008 (12) ◽  
pp. pdb.rec11574-pdb.rec11574
Keyword(s):  
Nuclear Isolation ◽  
Isolation Buffer

Association of a cGMP-binding activity with nuclei isolated from amoebae of Dictyostelium discoideum and Polysphondylium violaceum

1992 ◽  
Vol 70 (2) ◽  
pp. 169-173 ◽  
Author(s):  
Jeffrey R. Butler ◽  
M. Barrie Coukell
Keyword(s):  
Dictyostelium Discoideum ◽  
Cell Aggregation ◽  
Binding Activity ◽  
Dissociation Kinetics ◽  
Isolated Nuclei ◽  
Nuclear Isolation ◽  
Slow Dissociation ◽  
Isolation Buffer

When vegetative and early slug stage amoebae of Dictyostelium discoideum or Polysphondylium violaceum were lysed by filter breakage in a nuclear isolation buffer not containing detergents, substantial levels of a cGMP-binding activity with slow-dissociation kinetics were detected. After fractionation by centrifugation, 50% or more of this binding activity was associated with isolated nuclei. In addition, with Polysphondylium cells, the fraction of stable, nuclear-associated binding activity appeared to increase during cell aggregation. These results support the idea that cGMP might function in the nucleus during early development.Key words: cGMP-binding activity, nuclear isolation, Dictyostelium discoideum, Polysphondylium violaceum.

scholarly journals Nuclei Release Methods Comparison for Fresh Leaves of Rice (Oryza sativa) for Efficient High Throughput Flow Cytometry Ploidy Studies

2019 ◽  
Vol 8 (2) ◽  
pp. 31
Author(s):  
Isidre Hooghvorst ◽  
Xavier Serrat ◽  
Salvador Nogués
Keyword(s):  
Flow Cytometry ◽  
Dna Ploidy ◽  
Razor Blade ◽  
Bead Beating ◽  
Ploidy Determination ◽  
Buffer Solutions ◽  
Rice Leaves ◽  
Nuclear Isolation ◽  
First Time ◽  
Isolation Buffer

Flow cytometry trituration methods and the efficiency of isolation buffer solutions are compared in this study for extraction of nuclei from fresh leaves of rice. The razor blade sample trituration procedure has been widely used to release nuclei from tissues in many plant species, and combined with different isolation buffers for low throughput analysis. In contrast, the bead beating trituration method has rarely been used for DNA ploidy determination, despite it being proposed as a less tedious alternative procedure to prepare nuclear suspensions. In this study, bead beating was assessed and compared with the traditional chopping procedure. Each trituration method was combined with one of three nuclear isolation buffers (i.e. Hanson’s, Otto’s and LB01 buffer). Bead beating was applied for the first time using all three of the buffers, resulting in a rapid and effective procedure for ploidy determination in fresh rice leaves. In addition, bead beating saved, while reducing the exposure of the user to harmful substances. The best results were obtained when Hanson’s nuclear isolation buffer was combined with the bead beating trituration method.

scholarly journals POST-VACCINATED ALTERATIONS IN THE MARKERS OF LIPID AND PROTEIN OXIDATION IN THE GILLS OF RAINBOW TROUT (ONCORHYNCHUS MYKISS WALBAUM) IMMUNIZED AGAINST THE ENTERIC REDMOUTH DISEAS

2020 ◽  
pp. 24-35
Author(s):  
Halyna Tkachenko ◽  
Natalia Kurhaluk ◽  
Joanna Grudniewska ◽  
Agnieszka Pękala-Safińska
Keyword(s):  
Oxidative Stress ◽  
Lipid Peroxidation ◽  
Rainbow Trout ◽  
Oncorhynchus Mykiss ◽  
Oxidative Stress Biomarkers ◽  
Enzymatic Assays ◽  
Stress Biomarkers ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Derivatives Of ◽  
Isolation Buffer

The aim of the study was the evaluation of the content of oxidative stress biomarkers (2-thiobarbituric-acid-reacting substances as a biomarker of lipid peroxidation, aldehydic and ketonic derivatives of oxidatively modified proteins) in the gills of rainbow trout (Oncorhynchus mykissWalbaum) vaccinated by a vaccine against Yersiniaruckeri. Rainbow trout (Oncorhynchus mykiss Walbaum) with a mean body mass of (107.9±3.1) g were used in the experiments. The study was carried out in a Department of Salmonid Research, Inland Fisheries Institute in Rutki (Poland). Experiments were performed at a water temperature of 14.5±0.5°C and the pH was 7.5. The dissolved oxygen level was about 12 ppm with additional oxygen supply with a water flow of 25 L per min, a photoperiod of 12 hours per day. The fish were fed with a commercial pelleted diet at an optimal level, using 12-hour belt feeders for fish. All enzymatic assays were carried out at the Department of Zoology and Animal Physiology, Institute of Biology and Earth Sciences, Pomeranian University in Słupsk (Poland).The fish were kept for 60 days after vaccination at a water temperature of 14.5±0.5°C and pH 7.5. In our study, 15 rainbow trout from unhandled control and 15 vaccinated trout were used. Two months after immunization, samples from rainbow trout were collected. The fish were captured and killed 61 days post-vaccination (n = 15 in each group). Gills were removed in situ. The organs were rinsed clear of blood with cold isolation buffer and homogenized using a glass homogenizer H500 with a motor-driven pestle immersed in an ice water bath to yield a homogenate in proportion 1:9 (weight/volume). The isolation buffer contained 100 mMTris-HCl; a pH of 7.2 was adjusted with HCl. Homogenates were centrifuged at 3,000g for 15 min at 4°C. After centrifugation, the supernatant was collected and frozen at −20°C until analyzed. Protein contents were determined using the method of Bradford (1976) with bovine serum albumin as a standard. Absorbance was recorded at 595 nm. All enzymatic assays were carried out at 22±0.5°C using a Specol 11 spectrophotometer (Carl Zeiss Jena, Germany) in duplicate. The enzymatic reactions were started by the addition of the tissue supernatant. Our results demonstrated that immunization by the anti-Yersinia vaccine does not alter the gills of rainbow trout. Oxidative stress parameters examined in gills homogenate, i.e., lipid peroxidation as measured by the amount of TBARS, as well as aldehydic (increased by 18.9%) and ketonic derivatives of OMP (decreased by 6.5 %) were non-significantly changed (p>0.05) in gills of vaccinated fish. Thus, immunization by anti-Yersinia vaccine does not alter oxidative stress markers compared to unhandled control in the second month after immunization. Our results confirm that the vaccine against Y. ruckeri has no adverse effect on the condition and metabolism in the gills of the fish. Alterations in the content of oxidative stress biomarkers recorded in our studies are proof that the vaccine against Y. ruckeri has no negative effects.

Nuclear isolation and sequencing for mouse hypothalamus v1

2021 ◽  
Author(s):  
Zhengzheng Sophia Liang ◽  
Eric Vaughn ◽  
Dhananjay Bambah-Mukku ◽  
Catherine Dulac
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Brain Tissue ◽  
Sequencing Library ◽  
Nuclear Isolation

This protocol is intended for isolation of nuclei from fresh brain tissue in preparation for single-nuclei sequencing library using Chromium Single Cell 3’ Reagent Kits v2 or v3 (10X Genomics). For each step, tissue and reagents were kept on ice or at 4C, to reduce unwanted gene expression due to procedures and maintain nuclear integrity.

Subnuclear associations of the v-myb oncogene product and actin are dependent on ionic strength during nuclear isolation

1987 ◽  
Vol 7 (9) ◽  
pp. 3345-3348
Author(s):  
W J Boyle ◽  
M A Baluda
Keyword(s):  
Ionic Strength ◽  
Nuclear Structure ◽  
Direct Effect ◽  
Nuclear Matrix ◽  
Nuclear Actin ◽  
Hypotonic Treatment ◽  
Nuclear Isolation

The method used to isolate nuclei has a direct effect on the subnuclear association of the v-myb product, p48v-myb, and nuclear actin. Analysis of nuclei subjected to various isolation procedures showed that disruption of native nuclear structure during hypotonic treatment resulted in dissociation of p48v-myb from the nuclear matrix.

scholarly journals Early signalling mechanism in colonic epithelial cell response to gastrin

1995 ◽  
Vol 311 (3) ◽  
pp. 945-950 ◽  
Author(s):  
R R Yassin ◽  
K M Little
Keyword(s):  
Tyrosine Phosphorylation ◽  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Tyrosine Phosphatase ◽  
Promoting Effect ◽  
Kinase C ◽  
Growth Promoting ◽  
Colonic Cells ◽  
Signalling Cascade ◽  
Isolation Buffer

The hormone gastrin exerts a growth-promoting effect on gastrointestinal cells. The molecular mechanisms by which colonic epithelial cells respond to gastrin are still poorly understood. In this study, we demonstrate a novel feature of the action of gastrin on normal colonic cells, namely the rapid phosphorylation on tyrosine of phospholipase C gamma 1 (PLC gamma 1). Tyrosine phosphorylation of PLC gamma 1, elicited by gastrin, was transient, concentration-dependent, and was abrogated by pretreating the colonic cells with the gastrin-receptor antagonist proglumide, the tyrosine kinase inhibitor genistein, and by removal of the tyrosine phosphatase inhibitor orthovanadate from the isolation buffer. Tyrosine phosphorylation of PLC gamma 1 correlated with the time- and concentration-dependent decrease in the mass of membrane phosphatidylinositol 4,5-bisphosphate (PIP2) and the increase in the epithelial concentration of inositol 1,4,5-trisphosphate (IP3). Likewise, the stimulated increase in IP3 was also prevented by proglumide and genistein. Gastrin induced a definite but transient increase in the intracellular concentration of free Ca2+ [Ca2+]i, and increased membrane-translocation of immunoreactive alpha- and beta-protein kinase C. The data thus indicate that gastrin elicits at least one signalling cascade, through rapid tyrosine phosphorylation of PLC gamma 1, leading to the activation of a PIP2-specific PLC pathway.

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