nuclear Protocols

Category: Cell Biology and Cell Culture: Apoptosis Protocols: Apoptosis Analysis

Analysis of DNA Fragmentation Using the JAM Assay. By Shailaja Kasibhatla et al., The JAM assay is based on labeling nuclear DNA of cycling cells with [3H]thymidine and harvesting samples on glass fiber filters. Apoptosis will generate DNA fragments small enough to pass through the glass fiber filter, resulting in decreased radioactivity of the particular sample. Cell-mediated cytotoxicity or cell killing mediated by cytotoxic T lymphocytes (CTL) can also be measured by this technique. - [Read Analysis Of DNA Fragmentation Using The JAM Assay (Subscription Required)]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Includes staining protocols: Triple-Staining Adherent Cells with MitoTracker, Phalloidin,& Nuclear Dyes; Staining Adherent Cells with Cytokeratin Primary Antibodies & Synthetic Fluorophores; Staining Adherent Cells with Intermediate Filament Primary Antibodies & Synthetic Fluorophores; Staining Cells & Tissue Cryosections with Tubulin Primary Antibodies, Phallotoxins,& Synthetic Fluorophores; Triple-Staining Tissue Cryosections with Wheat Germ Agglutinin, Phalloidin,& Nuclear Dyes; etc. - [Read Confocal Microscopy Specimen Preparation Using Synthetic Fluorophores and Immunofluorescence]

Category: Cell Biology and Cell Culture: Apoptosis Protocols: NF-kB EMSA Protocols

Detailed NF-kB EMSA Protocol. Includes Nuclear extract preparation, Labeling of the oligonucleotide probe: T4 Polynucleotide Kinase Reaction, Supershift assay to identify NF-kB, and recipes for EMSA buffers. Celldeath.de - [Read Detailed NF-kB EMSA Protocol]

Category: DNA Protocols: DNA Extraction Blood

Purifying nuclear pellets, Extraction of DNA from nuclei. Method for DNA isolation from Blood. Genomic Variation Laboratory, UC Davis. - [Read DNA extraction from blood]

Category: DNA Protocols: EMSA DNA-Protein Protocols

Electrophoretic Mobility Shift Assay (EMSA) for the detection of nuclear NF-kB. CellDeath.de - [Read Electrophoretic Mobility Shift Assay (EMSA) for the detection of nuclear NF-kB]

Category: Staining Protocols: Gram Staining Protocols

Protocol for gram stain. Includes: Hucker-conn ammonium oxalate - Crystal violet; Weigerts Iodine; 0.1% Nuclear fast red. - [Read Gram Stain Protocol]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to distinguish mitochondrial mutations from nuclear mutations. - [Read How to Distinguish Mitochondrial Mutations from Nuclear Mutations]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Protocol for in vitro splicing reactions in Drosophila KC or HeLa nuclear extracts. Protocol includes: Procedure, Solutions, BioReagents and Chemicals and protocol hints. - [Read In Vitro Splicing Reactions in Drosophila KC or HeLa Nuclear Extracts]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

With this protocol, transcripts that were initiated from specific genes by RNA polymerases prior to permeabilization can be measured. Instead of a nuclear extract, permeabilized cells are used. Includes information on: Permeabilization of Cells; In vitro Transcription Reaction (Run-off); Isolation of RNA; Preparation of Slot Blot Membrane for Hybridization; Hybridization of Nitrocellulose Membrane; TCA Precipitation to Determine Incorporation of [32P] GTP into Nucleic Acid - [Read In Vitro Transcription Assay (Run-off Assay) using Permeabilized Cells]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Protocol for in vitro transcription with yeast nuclear extract. Includes recipes for: 5x Acetate transcription buffer; Creatine phospho kinase; Phospho creatine; Stop mix; HA + 0.1 M potassium acetate; 5x glutamate transcription buffer (5 ml). Includes also protocol for Primer Extension Assay. - [Read In Vitro Transcription With Yeast Nuclear Extract]

Category: Immunology: Immunofluorescence Protocols

The study of the activation of DNA damage response factors at the single cell level relies on the observation of their accumulation (or the accumulation of their activated forms) in nuclear foci by plain immunofluorescence techniques. - [Read Indirect Immunofluorescence with Preextraction (In Situ Cell Fractionation) Protocol]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

Protocol for the isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays. Plant materials are ground in hexylene glycol-based buffers and highly enriched nuclear fractions are obtained using Percoll density gradients. Standard and small-scale protocols are presented, along with a tested method for nuclear run-on assays. The entire process may be completed within 3 days. - [Read Isolation of Arabidopsis Nuclei and Measurement of Gene Transcription Rates Protocol]

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

Protocol for the isolation of genomic and viral DNA from lymphocytes. Includes: Viral DNA in cytosolic fraction and Viral DNA in nuclear fraction —integrated. - [Read Isolation of Genomic and Viral DNA from Lymphocytes Protocol]

Category: Protein Protocols: Nuclear Extraction Protocols

Homogenize cells or tissue, Pellet nuclei, Lyse nuclei, Reprecipitate nuclear proteins, dialyze nuclear extract. Hattori M et al., DNA Cell Biol. 1990. Homogenization buffer and dialysis buffer recipes. - [Read Large scale nuclear extract preparation]

Category: Cell Biology and Cell Culture: Apoptosis Protocols: Apoptosis Analysis

Leukostat Staining of Cytospin Preparations to Detect Apoptosis. Shailaja Kasibhatla et al. Leukostat staining is used to visualize nuclear changes and apoptotic body formation that are characteristic of apoptosis. Cells are viewed under a light microscope and counted to quantify apoptosis. This protocol can be used both for cells that grow in suspension and for adherent cells. - [Read Leukostat Staining of Cytospin Preparations to Detect Apoptosis]

Category: DNA Protein Interactions Protocols: Dnase Footprinting Assay Protocols

Dnase I is used to fragment a radiolabeled target DNA in the presence and absence of a nuclear extract.  A "footprint" is generated when a protein binds to the target and protects a specific segment of DNA from the nucleolytic activity of Dnase I. By comparing the electrophoretic mobility of the Dnase I cleavage products to those of a sequence ladder derived from the same DNA fragment, the position(s) of the DNA sequences recognized by DNA-binding proteins can be determined. - [Read Mapping Protein-binding Sites on DNA by Dnase I Footprinting Protocol]

Category: Protein Protocols: Nuclear Extraction Protocols

S100 extract cytosolic extraction preparation. Buffer A (Hypotonic Buffer), Low and High salt buffer recipes. Brent Graveley - [Read Nuclear and Splicing Extract Preparation]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Protocol describes a system which includes all of the necessary components for in vitro transcription as well as a positive control template that provides run-off transcripts from a CMV immediate early promoter. This system is designed for runoff transcription. Alternatively, transcription products can be analyzed by primer extension. - [Read Nuclear Extract in vitro Transcription System]

Category: DNA Protocols: EMSA DNA-Protein Protocols

Nuclear Extract Preparation for EMSA after Wadman et al., EMBO J. 1997. Buffer recipies included. - [Read Nuclear Extract Preparation for EMSA (PDF)]

Category: DNA Protein Interactions Protocols

Protocol for nuclear extract preparation. - [Read Nuclear Extract Preparation Protocol]

Category: Cell Organelle Protocols: Nucleus Protocols

Protocol for nuclear isolation. - [Read Nucleolar Isolation Protocol]

Category: Drosophila Protocols: Drosophila Genetics Protocols

Protocol describes how to produce a soluble nuclear extract rich in basal pol II transcription factors from Drosophila embryos. This is a cell-free extract that contains all the necessary transcription factors and is capable of accurate initiation of transcription by RNA polymerase II but is deficient in core histones and histone H1. - [Read Preparation of a Highly Efficient Transcription Extract from Drosophila Embryos Protocol]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Protocol for preparation of KC nuclear extract for in vitro splicing. Protocol makes 3.4 ml of extract for every 4 liter of cells (depending on initial cell concentration). Protocol includes: Procedure, Solutions, BioReagents and Chemicals and protocol hints. - [Read Preparation of KC Nuclear Extract for In Vitro Splicing]

Category: Protein Protocols: Nuclear Extraction Protocols

Nuclear Extract protocol is optimized for about 10^7 cells, can use for gel shift assays and western blotting. Mirmira Lab, Univ. Virginia - [Read Preparation of Nuclear Extracts for Gel Shifts and Westerns Blots]

Category: Cell Culture Protocols: Insect Cell Culture Protocols

Production of Autographa californica Multiple Capsid Nuclear Polyhedrosis Virus (rAcMNPV) Stock. - [Read Production of Autographa californica Multiple Capsid Nuclear Polyhedrosis Virus (rAcMNPV) Stock]