cross Protocols

Category: Genetics and Genomics: Genotyping Protocols: Mutation Detection Protocols

Describes an experimental cross in mice that can be used to define and map induced germ-line mutations that map to a single chromosome. The cross is a modification and extension of a conventional three-generation recessive mutagenesis screen. Includes: The Mutagenesis Breeding Plan; Consomic Strains; Generating Mutations; Generating and Genotyping G2 Females; Genotyping G3 Progeny; Phenotyping G4 Progeny; etc.. - [Read A Targeted Screen to Detect Recessive Mutations that have Quantitative Effects Protocol]

Category: C. elegans Protocols: C. elegans Staining Protocols

Extreme care should be used to identify and verify positive reactions, however, because cross-reactions are common. Counterstaining is essential for examining worms by immunofluorescence and is used to identify the exact cell in which an antigen appears. Methods for counterstaining include labeling all cells with a fluorescent dye that is specific for nucleic acids (e.g., DAPI or propidium iodide) and using GFP driven by tissue-specific promoters. - [Read Antibody Addition and Detection for Staining Caenorhabditis elegans Protocol]

Category: Immunohistochemistry Protocols: Blocking Protocols

Protocol for blocking of unwanted non-specific staining. Includes: Blocking of endogenous enzymes; Blocking of endogenous fluorochromes; Blocking endogenous biotin; Blocking of endogenous Fc blocking; Blocking of cross reactive antigens in the tissue. - [Read Blocking Unwanted Non-Specific Staining Protocol]

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

Formaldehyde cross-linking and chromatin immunoprecipitation assays of tissue culture cells, Based on Boyd and Farnham. Michelle Kallesen, Rosen Lab. - [Read ChIP Assay Protocol PDF]

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

Chromatin Immunoprecipitation Protocol for Microarray Analysis – Protein A/G Bead Method. Cross-Linking, Sonication/Chromatin Shearing Efficiency. Reaction UHN Microarray Centre - [Read Chromatin Immunoprecipitation Protocol PDF]

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

ChIP assay protocol with 2 steps: in vivo formaldehyde cross-linking of whole cells and protein-protein and protein-DNA interactions, followed by immunoprecipitation of protein-DNA complexes with specific antibodies from sonicated extracts. Breeden Lab - [Read Chromatin IP (CHIP assay)]

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

Mapping Protein/DNA Interactions by Cross-Linking Examining the Distribution of Telomeric and DNA Repair Proteins by ChrIP and Real-Time PCR - [Read Chromatin-IP (ChrIP) Protocol]

Category: Protein Protocols: Immunoprecipitation Protocols

Cross-linking and Immunoprecipitation Protocol. Molecular Station. - [Read Cross-linking and Immunoprecipitation Protocol]

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

Protocol describes the directed coupling of antibody to protein G-agarose via its Fc domain, and subsequent covalent cross-linking of the complex using dimethyl pimelimidate (DMP). - [Read Directed Orientation of Antibodies during Preparation of Antibody Affinity Resin Protocol]

Category: Cell Culture Protocols: Cell Fixing Protocols

Treating cells with paraformaldehyde leads to the establishment of chemical cross-links between free amino groups. When the cross-links join different molecules, a latticework of interactions occurs that holds the overall architecture of the cell together. Commercial formaldehyde solutions are not recommended, because they lack the advantages of using a variable-length polymer, and the cells will simultaneously be fixed with the alcohol (usually methanol). - [Read Fixing Attached Cells in Paraformaldehyde Protocol]

Category: Cell Culture Protocols: Cell Fixing Protocols

Treating cells with paraformaldehyde leads to the establishment of chemical cross-links between free amino groups. When the cross-links join different molecules, a latticework of interactions occurs that holds the overall architecture of the cell together. - [Read Fixing Suspension Cells with Paraformaldehyde Protocol]

Category: Protein Protocols: Immunoprecipitation Protocols

Information and tips on how to cross-link proteins. - [Read How to Cross-Link Proteins]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to make a cross. - [Read How to Make a Cross]

Category: Bacterial Protocols

Alkali is used to liberate DNA from bacterial colonies on nitrocellulose or nylon filters. The DNA is then fixed to the filter by UV-cross-linking or baking under vacuum. - [Read Lysing Colonies and Binding of DNA to Filters Protocol]

Category: Chromatography Protocols: Affinity Chromatography Protocols

Protocol describes the coupling of peptides or proteins to Affi-Gel 10 (Bio-Rad).  Affi-Gel 10 is a cross-linked agarose derivatized with N-hydroxysuccinimide (NHS) ester on a 10-atom-long spacer arm.  The NHS ester allows spontaneous coupling of proteins via free amino groups. - [Read Protein Ligand Affinity Chromatography Protocol]

Category: Signalling Signal Transduction Protocols

Protocol describes a competitive ligand binding assay for cortical neurotrophin receptors. Following binding in the presence of competitor, the bound radiolabeled ligand is cross-linked to the receptor. The cells are lysed and the ligand-receptor complexes are immunoprecipitated using a pan-trk (tyrosine kinase receptor) antibody. Protocol includes:Preparation of Cortical Tissue for Competitive Crosslinking, Competitive Binding, Crosslinking Ligand to Receptor, Lysis and Immunoprecipitation etc - [Read Protocol for Competitive Ligand Binding to Cortical Receptor using Crosslinking]

Category: Chromatography Protocols: Affinity Chromatography Protocols

Foreign proteins fused to maltose-binding protein can be readily purified to near homogeneity by affinity chromatography on resins containing cross-linked polysaccharides such as amylose. - [Read Purification of Maltose-binding Fusion Proteins by Affinity Chromatography on Amylose Resin Protocol]

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

Protocol describes a method for removing antibodies that react with bacterially encoded proteins by passing a crude preparation of immunoglobulins through a column containing immobilized bacterial proteins. - [Read Removal of Cross-reactive Antibodies from Antiserum: Affinity Chromatography Protocol]

Category: Antibody Protocols: Antibody Production Protocols

This protocol describes how antibodies that react with bacterial-encoded proteins may be removed from polyclonal antisera by incubation with a bacterial lysate. - [Read Removal of Cross-reactive Antibodies from Antiserum: Incubation with E. coli Lysate]

Category: Antibody Protocols: Antibody Production Protocols

Antibodies directed against bacterial and phage-encoded proteins can be removed from sera by incubation with nitrocellulose filters impregnated with lysates of uninfected or phage-infected E. coli. - [Read Removal of Cross-reactive Antibodies from Antiserum: Pseudoscreening Protocol]

Category: RNA Protocols: RNA-binding Protein EMSA Interactions

SULLY et al., 2004 Biochem. J. 377 (629–639). - [Read RNA EMSA (electrophoretic mobility shift assays) and UV cross-linking]

Category: Neuroscience Protocols: Neuronal Stains Protocols: Stains for Senile Plaques Protocols

Protocol for senile plaques and neurofibrillary tangles. - [Read Senile Plaques and Neurofibrillary Tangles Protocol]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

Arabidopsis naturally self-pollinates, the generation of cross-progeny requires some intervention by the investigator. This protocol describes the generation and collection of seeds by crossing suitable Arabidopsis parent plants. - [Read Setting Up Arabidopsis Crosses Protocol]

Category: DNA Protein Interactions Protocols

The multiprotein-DNA complex of interest is formed using the site-specifically derivatized DNA fragment.  The complex is then UV-irradiated, initiating covalent cross-linking with proteins in direct physical proximity to the cross-linking agent.  Extensive nuclease digestion is performed to eliminate uncross-linked DNA and convert cross-linked DNA to a cross-linked, radiolabeled nucleotide "tag." - [Read Site-Specific Protein-DNA Photo-Cross-Linking: Analysis of Structural Organization of Protein-DNA]

Category: Cell Culture Protocols: Trypsinization Protocols

Protocol describes the trypsinization of cells in monolayer culture to facilitate subculture or harvesting. To avoid cross-contamination of cells, it is important for each cell line to be subcultured independently. No more than one cell line should be in the tissue culture hood at any one time. - [Read Trypsinization of Cells Grown in Monolayer Protocol]