nuclear Protocols

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: 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: 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: 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: 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: 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: 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: 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: Protein Protocols: Nuclear Extraction Protocols

Nuclear Extract preparation protocols, Buffers and solutions for nuclear extracts. Hahn Lab - [Read Yeast Nuclear Extract (Small Scale)]

Category: Yeast Protocols: Yeast Staining Protocols

Protocol describes the staining of nuclear and mitochondrial DNA with DAPI (4',6-diamidino-2-phenylindole). - [Read Yeast Vital Stains: DAPI Stain of Nuclear and Mitochondrial DNA Protocol]

Category: Cell Culture Protocols: Insect Cell Culture Protocols

Includes protocols: Direct Adaptation to HyQ© SFX-Insect™ Medium; Sequential Adaptation to HyQ© SFX-Insect™ Medium; Adaptation of High Five™ Cells to HyQ© SFX-Insect™; Production of Recombinant Protein Using the Baculovirus Expression Vector System; Culturing Insect Cells and Production of Baculovirus Expressed Proteins in 2.8 L Fernbach Systems. Production of Autographa californica Multiple Capsid Nuclear Polyhedrosis Virus (rAcMNPV) Stock - [Read Protocols for Insect Cell Culture]

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 Organelle Protocols: Nucleus Protocols

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

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: 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: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

GUS is used as a tag to address nuclear localization whereas GFP is more versatile. GFP is detectable directly in living cells, GUS is only detected indirectly by staining of fixed tissue which may lead to artifacts or may obscure problems with protein solubility. In this protocol, protein localization is routinely assayed after particle-mediated transient transformation of onion epidermal cells. With this method it can be determined rapidly whether a given fusion protein is active and.... - [Read Subcellular Localization of GUS- and GFP-Tagged Proteins in Onion Epidermal Cells]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

This protocol describes nuclear and cytoplasmic fractionation of tissue culture cells and a method for Western blot detection of proteins using the Odyssey Infrared Imaging System. This protocol was used to detect expression of the "small" Tap protein in 293T, HeLa and COS cells. The Odyssey system has several advantages over the more widely used chemiluminescent detection methods. - [Read Western Blot Analysis of Sub-Cellular Fractionated Samples Using the Odyssey Infrared Imaging System]

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: 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: DNA Protein Interactions Protocols

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

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: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes a useful way to observe the development of embryos, as well as meristems & young primordia developing at the shoot apex by confocal microscopy after staining the nuclei with propidium iodide. The number of cells can be exactly quantified in a meristem or in young primordia. Because embryonic & meristematic cells are largely filled out by their nuclei, it is easier to image only the nuclei. This method allows analysis of whole-mount material, which is more easily reconstructed. - [Read Protocol for Nuclear Staining of Plants for Confocal Microscopy]

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: 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: Developmental Biology: Embryology Protocols

Protocol to Count Cell Number of Preimplantation Embryos using Nuclear Staining with Hoechst 33342 or DAPI. Includes: Preparation of Embryos; Preparation of Hoechst 33342 dye; Preparation of DAPI; Staining the Embryo; Mounting Embryos to Slides; What to Do When There are Too Many Cells to Count. - [Read Protocol to Count Cell Number of Preimplantation Embryos]