interactions Protocols

Category: Molecular Biology Protocols: Carbohydrate Protocols

High performance liquid affinity chromatography (HPLAC) is a useful procedure to investigate he interactions between carbohydrate binding protein and their ligands. Technical requirements are similar to conventional HPLC. HPLAC can screen and separate natural ligands from complex biological mixtures. WeiTong Wang~GlycoTech Corporation, Rockville, Maryland - [Read Analysis of Oligosaccharide Ligands by High Performance Liquid Affinity Chromatography]

Category: Fungi Protocols

Serum concentrations of itraconazole should be measured in patients receiving this drug to ensure that therapeutic concentrations are being achieved. This is necessary as drug absorption can be variable, and levels may be lowered by interactions with other drugs. The assay will give an indication of whether suitable blood levels have been achieved. - [Read Bioassay for Determining Itraconazole Levels in Blood]

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

BN-PAGE has become the method of choice for the investigation of the respiratory protein complexes of the electron transfer chains of a range of organisms. It allows the separation in two dimensions of extremely hydrophobic protein sets for analysis and also provides information on their native interactions. In this review we discuss the capabilities of BN-PAGE in proteomics and the wider investigation of protein:protein interactions with a focus on its use and potential in plant science. - [Read Blue-Native PAGE in Plants: A Tool in Analysis of Protein-Protein Interactions]

Category: Immunology: Immunoprecipitation Protocols: Chromatin Immunoprecipitation Protocols

Protocol describes the use of chromatin immunoprecipitation technology (ChIP) to analyze interactions of proteins or protein complexes with DNA in vivo. In this approach, the material is fixed with formaldehyde to preserve DNA-protein and protein-protein associations, the cells are lysed, and the chromatin is cut and solubilized. The chromatin suspension is immunoprecipitated with an antibody against the protein(s) of interest, and the coimmunoprecipitated DNA fragments are analyzed. - [Read Chromatin Immunoprecipitation (ChIP) of Protein Complexes Protocol]

Category: Immunology: Immunoprecipitation Protocols: Chromatin Immunoprecipitation Protocols

Genome-wide location analysis, also known as ChIP-Chip, combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in living cells. Protein-DNA interactions are captured in vivo by chemical crosslinking. Cell lysis, DNA fragmentation and immunoaffinity purification of the desired protein will co-purify DNA fragments that are associated with that protein. - [Read Chromatin Immunoprecipitation and Microarray-Based Analysis of Protein Location Protocol]

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: Co-immunoprecipitation

Co-immunoprecipitation Analysis of Protein-Protein Interactions. Morrisey Lab - [Read Co-immunoprecipitation Analysis of Protein-Protein Interactions]

Category: Transfection Protocols: Stable Transfection Protocols

CHO Lec3.2.8.1 cells have 4 independent mutations in the N and O glycosylation pathways. When cultured with alpha-glucosidase I inhibitor N-butyl-deoxynojirimycin, glycoproteins produced in CHO Lec3.2.8.1 cells are completely susceptible to Endo H digestion. Endo H cleaves chitobiose, leaving a single N-linked N-acetylglucosamine per site which is ideal for maintenance of protein solubility and special carb-protein interactions, such as between the first N-acetyl glucosamine residue and tryp. - [Read Establishment of Stable Transfectant of CHO Lec Cells 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: Yeast Protocols: Yeast Protein Protocols: Yeast Protein Protein Interactions Protocols

This procedure capitalizes on the asexual/sexual reproductive cycle of yeast cells. Laboratory yeast strains can be maintained indefinitely by asexual reproduction as one of two haploid mating types, MATa or MAT{alpha}. - [Read Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array]

Category: Yeast Protocols: Yeast Protein Protocols: Yeast Protein Protein Interactions Protocols

This protocol describes how to handle arrayed yeast using robotics. - [Read Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array:]

Category: Yeast Protocols: Yeast Protein Protocols: Yeast Protein Protein Interactions Protocols

This protocol describes the first step in constructing an array: amplification of the predicted ORFs that are to be included in the array. Gene-specific primers containing vector-specific flanking sequences that facilitate recombinational cloning are used to amplify each ORF. A secondary amplification can be used to extend the length of the homologous vector sequence flanking the ORF. - [Read Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array: Amplification of ORFs]

Category: Yeast Protocols: Yeast Protein Protocols: Yeast Protein Protein Interactions Protocols

This protocol describes three different storage methods for the array. Frozen stocks are the most permanent method and are an absolute requirement for maintaining a record of the array. - [Read Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array: S]

Category: Immunology: Immunoprecipitation Protocols: Immunoprecipitation of Radiolabeled Cells Protocols

Coimmunoprecipitation is most commonly used to test whether two proteins of interest are associated in vivo, but it can also be used to identify novel interacting partners of a target protein. In both cases, the cells, which may have been labeled with [35S]methionine, are harvested and lysed under conditions that preserve protein-protein interactions. The target protein is specifically immunoprecipitated from the cell extracts, and the immunoprecipitates are fractionated by SDS-PAGE. - [Read Identification of Associated Proteins by Coimmunoprecipitation Protocol]

Category: Western Blot Protocols

Protein immunoprecipitation can be a useful preparative step for immunoblotting.  For very rare proteins, the protein of interest can be purified and concentrated by standard immunoprecipitation techniques before immunoblotting.  In addition, protein-protein interactions can be tested with an immunoprecipitating antibody that is specific for one protein of a complex and an immunoblotting antibody that is specific for a second member of the complex. - [Read Immunoblotting: Preparing Immunoprecipitated Proteins Protocol]

Category: Immunology: T Cell Protocols

Describes generating CTL against some commonly used target antigens. Two methods for the quantitation of CTL activity are described based on the two pathways used bt CTL to kill target cells. In one pathway, they release lytic granules containing perforin and granzymes, leading to apoptosis and target cell lysis. In a second pathway, they trigger apoptosis via Fas/Fas ligand interactions. - [Read Induction and Measurement of Cytotoxic T Lymphocyte Activity Protocol]

Category: Cell Culture Protocols: Cell Lysis Protocols

For cells grown in tissue culture, the most useful method of lysis is treating with detergents, as described in this protocol. Non-ionic detergents, such as NP-40, solubilize the plasma and intracellular membranes, break many weak intermolecular bonds, and solubilize most of the commonly studied protein antigens. RIPA lysis buffer may be used as a more rigorous extraction buffer to release all but the insoluble proteins of the cell and to break most weak noncovalent interactions. - [Read Lysing Tissue-Culture Cells for Immunoprecipitation Protocol]

Category: Reporter Gene Assays: GFP Assay Protocols

Protocol specifically describes data acquisition for a particular variant of GFP (EGFP) or Oregon Green as a donor fluorophore, but it can be adapted for image acquisition of other chromophore. - [Read Probing Protein Interactions Using GFP and FRET]

Category: Reporter Gene Assays: GFP Assay Protocols

In preparation for FLIM-FRET analysis, the appropriate donor and acceptor components must be introduced into live or fixed cells. The method of introduction depends on the nature of the components and the state of the cells. For example, plasmid DNAs encoding a protein of interest fused to a variant of GFP may be introduced into live cells by transfection or microinjection, whereas labeled antibodies are delivered by microinjection. - [Read Probing Protein Interactions Using GFP and FRET Protocol]

Category: Reporter Gene Assays: GFP Assay Protocols

Describes how components (usually Fab fragments of antibodies) that are to be introduced into cells are labeled with fluorescent sulfoindocyanine dyes. - [Read Probing Protein Interactions Using GFP and FRET.]

Category: Plant Biology Protocols: Arabidopsis Protein Peptide Protocols

Coimmunoprecipitation is one of the most useful techniques for revealing protein-protein interactions. Good negative controls to verify the specificity of the coimmunoprecipitation procedure are (1) performing the same immunoprecipitation experiment using beads coupled to the preimmune serum and (2) probing the Western blot with antibodies against protein known not to interact with the coimmunoprecipitated proteins under physiological conditions. - [Read Protein Coimmunoprecipitation in Arabidopsis Protocol]

Category: Protein Microarray Protocols

Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions. Gobom. - [Read Protein microarray technology and ultraviolet crosslinking combined]

Category: Cell Biology and Cell Culture

This protocol focuses on the interactions between L-selectin expressed on neutrophils and PNAd coated onto the plastic surface. The main purpose of the flow chamber assay is to visualize and measure interactions between flowing cells expressing a given adhesion molecule on their surface, and their receptor, either directly coated on the flow chamber lower wall or expressed on a cell monolayer. - [Read Protocol for L-selectin-PNAd Interactions under Flow Conditions.]