ligand Protocols

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Intracellular Ion Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium (Ca2+) in cultured RAW 264.7 cells. This objective is accomplished with the Ca2+-sensitive fluorescent dye, fluo-3, which permeates cells as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. Fluorescence is measured over time with adherent cells that have been washed free of extracellular dye. - [Read Assay of Intracellular Free Calcium in RAW 264.7 Cells for Ligand Screen Protocol]

Category: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

The following protocol is a method to measure concentrations of free cytoplasmic calcium (Ca2+) in mouse splenic B cells in the presence of two ligands. Signalling Gateway - [Read Assay of Intracellular Free Calcium in Suspended B Cells Dual Ligand PDF]

Category: Gene Delivery Protocols: DNA Nanoparticles Protocols

This protocol describes a stepwise procedure to prepare nucleic acids encapsulated in a polyethylene glycol (PEG)-shielded nanolipoparticle (NLP) that contain a bioresponsive lipid and ligand. This process provides several advantages for systemic gene delivery. The in vivo circulation time is extended. Also, low pH-sensitive lipids enhance DNA unpacking and endosomal escape. Finally, ligands inserted into the NLP surface can target gene delivery to specific tissues or cells in vivo. - [Read Bioresponsive Targeted Charge Neutral Lipid Vesicles for Systemic Gene Delivery 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: Protein Protocols: Protein Phosphorylation Protocols

A powerpoint slide demonstrating a method for Monitoring Protein Phosphorylation for Ligand Screens. Mumby. signaling-gateway.org - [Read Monitoring Protein Phosphorylation for the Ligand Screens PPT]

Category: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Protocol for the optimization of absorption condition for dye-ligand affinity chromotography. Generally, low pH and low ionic strength, absence of phosphate ions, and the presence of divalent metals ions increase the binding of proteins to immobilized triazine dyes. - [Read Optimization of Adsorption Conditions for Dye-Ligand Affinity Chromatography Protocol]

Category: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Protocol for the optimization of elution condition for dye-ligand affinity chromotography. Elution methods used in dye-ligand affinity chromatography may be either selective or nonselective in nature.  Usually, selective elution methods are applied in combination with group-specific adsorbents, such as dye-ligand adsorbents, and nonselective elution methods are used in combination with highly specific adsorbents. - [Read Optimization of Elution Conditions for Dye-Ligand Affinity Chromatography Protocol]

Category: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Triazine dyes, such as Cibacron Blue 3GA, can be linked to a hexyl spacer arm and then immobilized on a polyhydroxyl support matrix that has been activated with either 1,1-carbonyldiimidazole or epichlorohydrin.  An alternative procedure for immobilizing dyes using the direct coupling method is provided in Immobilization of Dyes on Polyhydroxyl Matrices Using the Direct Coupling Method. - [Read Preparation of Affinity-Ligand Resins by Immobilization of Dyes on Polyhydroxyl Matrices]

Category: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Protocol describes a general approach for the direct attachment of a triazine dye onto a carbohydrate support.  The direct coupling method is achieved under alkaline conditions (pH 10-11) with nucleophilic displacement of the dye’s chlorine atom by the polymer hydroxyls. - [Read Preparation of Affinity-Ligand Resins by Immobilization of Dyes on Polyhydroxyl Matrices Protocol]

Category: Signalling Signal Transduction Protocols

Protocol provides a method of achieving a sufficient sample for several determinations of cAMP using Amersham’s acetylation protocol. Protocol includes information on: Treatment of Cells and Preparation of Extracts; Reagents and Materials. - [Read Preparation of B-Lymphocyte Lysates for Cyclic AMP Determination for Dual Ligand Screen]

Category: Immunology: B Cell Protocols

The method chosen for measuring the content of cyclic adenosine 3',5'- monophosphate (cyclic AMP or cAMP) in splenic B lymphocytes (B cells) is an enzymelinked immunoassay system. Includes: Treatment of Cells and Preparation of Extracts. - [Read Preparation of B-Lymphocyte Lysates for Cyclic AMP Determination for Dual Ligand Screen 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: Dye Ligand Affinity Chromatography Protocols

Protocol describes a general method for the purification of protein using dye-ligand affinity chromatography.  The effectiveness of this protocol is dependent upon the reader having previously determined the optimal dye and buffer conditions to use for binding and eluting the protein(s) of interest. - [Read Purification of Protein Using Dye-Ligand Affinity Chromatography Protocol]