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Affinity Purification of Interacting Proteins from Cell Lysates Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/3/pdb.prot4593

Category: Chromatography Protocols: Affinity Chromatography Protocols

Recombinant protein or a chemically synthesized bioactive fragment is immobilized on resin and used as a probe to capture interacting proteins directly from a cell extract. Affinity-purified proteins are fractionated by gel electrophoresis and visualized by Coomassie staining. Proteins that interact specifically are identified by comparing this gel profile to one obtained from cell lysates passed over a control resin lacking the immobilized probe protein. - [Read Affinity Purification of Interacting Proteins from Cell Lysates Protocol]

Calcium Dependent by Pentraxins to Ligands, and Effect of C-reactive Protein on Leptin Action - http://www.natureprotocols.com/2007/01/24/calcium_dependent_by_pentraxin.php

Category: Mouse Protocols

Protocol for calcium dependent by pentraxins to ligands immobilized on agarose, and effect of C-reactive protein on leptin action in mice in vivo. Includes: Testing for binding of CRP to immobilized leptin; Testing for binding of radiolabelled leptin by immobilized CRP, SAP and leptin receptor; Testing for effect of human CRP on human leptin action in mice. - [Read Calcium Dependent by Pentraxins to Ligands, and Effect of C-reactive Protein on Leptin Action]

Chelating Sepharose Fast Flow Protocol - http://www.bio.indiana.edu/~ybelab/procedures/ChelatingSepharose.pdf

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Immobilized metal ion affinity chromatography (IMAC) exploits a molecule’s affinity for chelated metal ions. The amino acid histidine present in many proteins forms complexes with transition metal ions such as Cu2+, Zn2+, Ni2+ and Fe3+. Chelating Sepharose™ Fast Flow with a suitable immobilized metal ion will therefore selectively retain proteins with exposed histidine. - [Read Chelating Sepharose Fast Flow Protocol]

Construction of Micro-Tip for Use in IMAC Protocol - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000163.pdf?pid=PP00000163

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol is the second step in a three-step process for the preparation and enrichment of phosphopeptides using immobilized metal affinity chromatography (IMAC) for the identification of the phosphopeptides by LCMS/ MS. This procedure describes the construction of microchromatographic columns, or micro-tips. - [Read Construction of Micro-Tip for Use in IMAC Protocol]

Fractionation of Maize Embryo Proteins for 2-D Gel Electrophoresis Using Multicompartment Electrolyz - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4235

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

Protocol describes a method for performing isoelectric fractionation of a maize embryo sample using a multicompartment electrolyzer(MCE). This prefractionation of proteins having pIs within a certain pH interval is essential for allowing high loads of protein to be resolved on narrow and ultra-narrow immobilized pH gradients used in 2D electrophoresis. The isoelectric membranes in the MCE act like isoelectric traps capturing all the protein species having pIs encompassing the pI value of each... - [Read Fractionation of Maize Embryo Proteins for 2-D Gel Electrophoresis Using Multicompartment Electrolyz]

Identifying DNA-binding Proteins in Bacteriophage ambda Expression Libraries Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3785

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Protocol describes how to identify cloned cDNAs encoding proteins that bind to specific DNA sequences. The methods used are very similar to those used for immunological screening of expression libraries except that the nitrocellulose filters carrying immobilized proteins are screened with 32P-labeled double-stranded DNA rather than with antibodies. - [Read Identifying DNA-binding Proteins in Bacteriophage ambda Expression Libraries Protocol]

Immunoblotting: Antigen Detection Using Chemiluminescence Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4271

Category: Western Blot Protocols: Antigen Detection Methods Protocols

The blot is blocked to prevent nonspecific adsorption of the immunological reagents. Antibodies are then bound to the proteins immobilized on the membrane, and the antigen is detected by labeling the antibodies with conveniently identified tags. Common labeling methods for chemiluminescent detection include anti-immunoglobulin antibody-coupled enzymes such as horseradish peroxidase, which catalyzes the oxidation of luminol and in turn releases light. - [Read Immunoblotting: Antigen Detection Using Chemiluminescence Protocol]

Immunoblotting: Antigen Detection Using Chromogenic Methods Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4272

Category: Western Blot Protocols: Antigen Detection Methods Protocols

Lysis and Protein Extraction from WEHI-231 Cells with TriPure Isolation Reagent Protocol - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000162.pdf?pid=PP00000162

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol is the first step in a three-step process for the preparation and enrichment of phosphopeptides using immobilized metal affinity chromatography (IMAC) for the identification of the phosphopeptides by LC-MS/MS. This procedure is used to prepare protein extracts from WEHI-231 cells. This preparation method provides total cellular protein samples that are free of contaminating nucleic acids. - [Read Lysis and Protein Extraction from WEHI-231 Cells with TriPure Isolation Reagent Protocol]

Measuring Protein Concentration by Western Analysis Using Enhanced Chemiluminescence Detection - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4255

Category: Western Blot Protocols: Antigen Detection Methods Protocols

Protocol describes, samples containing the target protein are deposited onto a polyvinyldifluoride (PVDF) membrane using a vacuum manifold. The immobilized protein is exposed to an antibody specific for the target protein, followed by an antibody that reacts with species-specific determinants carried by the primary antibody and is conjugated to horseradish peroxidase (HRP). - [Read Measuring Protein Concentration by Western Analysis Using Enhanced Chemiluminescence Detection]

Optimization of Adsorption Conditions for Dye-Ligand Affinity Chromatography Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4210

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]

Optimization of Imidazole Concentrations for Immobilized Metal-Ion Affinity Chromatography Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/9/pdb.prot4261

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol for the optimization of imidazole concentrations for immobilized metal-ion affinity chromatography. Most samples from which histidine-tagged proteins are to be purified also contain endogenous protein contaminants that bind to the immobilized metal-ion affinity chromatography (IMAC) adsorbent. Usually, these proteins bind more weakly than the histidine-tagged protein. - [Read Optimization of Imidazole Concentrations for Immobilized Metal-Ion Affinity Chromatography Protocol]

Packing an IMAC Column Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4215

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol for packing an IMAC column. Protocol describes a procedure for packing a 30-ml immobilized metal-ion affinity chromatography (IMAC) column with the aid of a pump. - [Read Packing an IMAC Column Protocol]

Preparation and Enrichment of Phosphopeptides from Phosphotyrosine Protocol - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000193.pdf?pid=PP00000193

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol is specifically for the further enrichment of phosphopeptides from a phosphotyrosine pull-down. This is the final step for the preparation and enrichment of phosphopeptides using immobilized metal affinity chromatography (IMAC) for the identification of the phosphopeptides by liquid chromatography tandem mass spectrometry (LC-MS/MS). - [Read Preparation and Enrichment of Phosphopeptides from Phosphotyrosine Protocol]

Preparation and Enrichment of Phosphopeptides Using IMAC and LC-MS/MS - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000164.pdf?pid=PP00000164

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol is the third step in a three-step process for the preparation and enrichment of phosphopeptides using immobilized metal affinity chromatography (IMAC) for the identification of the phosphopeptides by LCMS/ MS. - [Read Preparation and Enrichment of Phosphopeptides Using IMAC and LC-MS/MS]

Preparation of a Prepacked IMAC Column Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4214

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol for the preparation of a prepacked IMAC column. Purification of histidine-tagged proteins on a milligram scale can be performed conveniently on a small (1to 5-ml) immobilized metal-ion chromatography (IMAC) column using a syringe to load the sample, wash the column, and elute the protein. - [Read Preparation of a Prepacked IMAC Column Protocol]

Preparation of Affinity-Ligand Resins by Immobilization of Dyes on Polyhydroxyl Matrices - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4208

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]

Protein Purification Using Immobilized Metal-Ion Affinity Chromatography under Optimized Imidazole - http://www.cshprotocols.org/cgi/content/extract/2006/9/pdb.prot4262

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Perform this protocol after determining the imidazole concentration that yields the best purification of the recombinant protein of interest. - [Read Protein Purification Using Immobilized Metal-Ion Affinity Chromatography under Optimized Imidazole]

Protocol for Transcriptional Run- On Assays - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3956

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

In this protocol nuclei isolated from cells expressing the gene of interest are incubated with radiolabeled UTP which is incorporated into nascent RNA transcripts by RNA polymerase molecules that were actively transcribing at the time the cells were harvested. Because very little denovo initiation of RNA synthesis occurs in isolated nuclei, transcription of the target gene can be measured by hybridizing the radiolabeled RNA to an excess of the target gene immobilized on a nitrocellulose or nylon - [Read Protocol for Transcriptional Run- On Assays]

Purification of Histidine-tagged Proteins by Immobilized Ni2+ Absorption Chromatography Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4088

Category: Chromatography Protocols: Affinity Chromatography Protocols

Recombinant proteins engineered to have a polyhistidine tail at either the carboxyl or amino terminus can easily be purified in one step by affinity chromatography on a resin carrying chelated nickel ions. Chromatography can be carried out in column or batch formats. After unbound proteins are washed away, the target protein is eluted using imidazole, which typically preserves the antigenic and functional features of the protein. - [Read Purification of Histidine-tagged Proteins by Immobilized Ni2+ Absorption Chromatography Protocol]

Purification of Histidine-Tagged Proteins under Denaturing Conditions Using IMAC Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4221

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Immobilized metal-ion affinity chromatography (IMAC) is suitable for the purification of proteins under denaturing conditions. Either guanidine-HCl or urea can be used, although guanidine-HCl is a stronger denaturant than urea. Proteins that have been adsorbed to the column in the presence of guanidine-binding buffer may be washed with urea-binding buffer and eluted with urea elution buffer. - [Read Purification of Histidine-Tagged Proteins under Denaturing Conditions Using IMAC Protocol]

Purification of Histidine-Tagged Proteins Using IMAC Without Parameter Optimization Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4217

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Histidine-tagged proteins can be purified on prepacked 1-ml immobilized metal-ion affinity chromatography (IMAC) columns without optimization of the separation conditions. The method allows fast capture of the target protein, although with a lower purity than can be obtained under optimized conditions. - [Read Purification of Histidine-Tagged Proteins Using IMAC Without Parameter Optimization Protocol]

Removal of Cross-reactive Antibodies from Antiserum: Affinity Chromatography Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3340

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]

Screening a cDNA Library for use with HybriZAP zebrafish cDNA libraries - http://www.cbs.umn.edu/~kclark/protocols/library.html

Category: RNA Protocols: cDNA Libraries Library

Procedures: Titer the library to determine the concentration of infectious phage, Plate and lift the phage particles, Hybridize the immobilized cDNA with labeled probe, Pick positive plaques. Karl Clark U. Minn. - [Read Screening a cDNA Library for use with HybriZAP zebrafish cDNA libraries]

Screening Immobilized Dyes for their Ability to Bind a Target Protein Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4209

Category: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Protocol for screening immobilized dyes for their ability to bind a target protein. The selection of a dye adsorbent for the purification of the target protein is an empirical undertaking and is generally achieved by a trial-and-error approach. - [Read Screening Immobilized Dyes for their Ability to Bind a Target Protein Protocol]

Articles (1-1 of 1)

Immobilized Antigen Phage Antibody Selection Protocol

A protocol for the selection of Phage Antibodies using Immobilized Antigen. This method describes the selection of antibodies from bacteriophage antibody libraries that recognize a specific antigen. The phage display library of antibody-displaying phage particles is exposed to antigen attached to a solid substrate (Nunc Immuno™ tubes). The phage particles with affinity for antigen bind to the immobilized antigen and are selected from the library of phage expressing antibodies.

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