antigen Protocols

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Protocol for estimating the number of CD38 molecules on the CD8+ T lymphocytes of HIV-infected individuals. Includes: RECOMMENDATION OF VENDOR FOR PE-CD38 AND PE-CD4; VALIDATION OF LOGARITHMIC AMPLIFIER LINEARITY AND SENSITIVITY; CONSERVATION OF THE LEVEL OF CD4 ANTIGEN EXPRESSION ON CD4+ LYMPHOCYTES AND ITS USE AS A BIOLOGIC STANDARD FOR FLOW CYTOMETER INSTRUMENT CHARACTERIZATION; DETERMINATION OF THE NUMBER OF CD38 MOLECULES PER CD8+ CELL; etc.. - [Read Estimating the Number of CD38 Molecules on the CD8+ T Lymphocytes of HIV-Infected Individuals]

Category: C. elegans Protocols: C. elegans Fixing Protocols

Parameters for fixation and staining. Includes: Antigen, Fixation, Fixation time, Antibody and Dilution. - [Read Fixation and Staining Parameters]

Category: Cell Culture Protocols: Cell Fixing Protocols

Organic solvents such as alcohols and acetone remove lipids and dehydrate cells, precipitating the proteins on the cellular architecture. Be aware that different antigens may be affected differently by the various solvents. If no previous data are available for your antigen, start with the 50/50 mixture. For tissue culture dishes, concentrations of acetone higher than 50% will destroy the integrity of the plastic. - [Read Fixing Attached Cells in Organic Solvents Protocol]

Category: C. elegans Protocols: C. elegans Fixing Protocols

Bouin’s fixative is a particularly good choice for worms because it penetrates dense tissues well and is extremely good for fixing antigens. Like all strong fixatives, however, it is unsuitable for some antibody-antigen pairs. In such cases, the length of time in the Bouin’s fixative can be shortened, or paraformaldehyde fixation can be used instead. - [Read Fixing Caenorhabditis elegans in Bouin’s Fixative Protocol]

Category: Molecular Imaging: Immunofluorescence Microscopy

General Immunofluorescence Protocol. Rosen Lab. Deparaffinize and rehydrate sections, Antigen retrieval methods, Proteinase K Antigen Retrieval, Urea Antigen Retrieval, Blocking buffers, - [Read General Immunofluorescence Protocol]

Category: Immunohistochemistry Protocols

Protocol for general immunohistochemistry. Includes: Antigen retrieval methods; Sodium Citrate Antigen Retrieval; Proteinase K Antigen Retrieval; Urea Antigen Retrieval. - [Read General Immunohistochemistry Protocol]

Category: ELISA Protocols

Protocol for HIV-1 p24 antigen capture ELISA. - [Read HIV-1 p24 Antigen Capture ELISA Protocol]

Category: Viral Protocols

Protocol for HIV-1 p24 antigen capture ELISA. - [Read HIV-1 p24 Antigen Capture ELISA Protocol]

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

This elution procedure relies on disrupting the bonds between the antibody and the antigen. - [Read Immunoaffinity Purification: Eluting Antigens from Immunoaffinity Columns Protocol]

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]

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. - [Read Immunoblotting: Antigen Detection Using Chromogenic Methods Protocol]

Category: Western Blot Protocols: General Western Blot Methods Protocols

For immunoblotting experiments, it is often important to compare the total amount of an antigen from many different sources or to learn if a particular source has the antigen under study.  In the approach described here, tissue cultures, bacteria, yeast cells, tissues, and other sources of antigens are disrupted directly in an electrophoresis sample . - [Read Immunoblotting: Preparing Cell Lysates Protocol]

Category: Immunohistochemistry Protocols

Protocol describes the application of peroxidase or alkaline phosphatase conjugates in the immunohistochemical labeling of formalin-fixed, paraffin-embedded tissue sections. Includes: Removal of Paraffin and Rehydration; Antigen Retrieval - Unmasking of Antigen; Enzyme retrieval; Microwave retrieval; Inactivation of Endogenous Peroxidase; etc.. - [Read Immunohistochemistry Protocol]

Category: Protein Protocols: Immunoprecipitation Protocols: Immunoprecipitation Troubleshooting

Immunoprecipitation Troubleshooting Guide. Stressgen. Problem: Non-specific background, Specific Background, Specific antigen not detected. With Solutions to common IP problems. - [Read Immunoprecipitation Troubleshooting Guide PDF]

Category: Immunology: Immunoprecipitation Protocols

For many sources of antigens, one useful method of lysis is to treat cells with harsh, denaturing solutions to release most of the protein antigens, as described here. The lysates are then diluted to reduce the denaturing conditions to levels that are suitable for the formation of antibody-antigen complexes. The resulting solution is precleared prior to immunoprecipitation. - [Read Immunoprecipitation: Denaturing Lysis Protocol]

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

To reduce backgrounds and to improve the signal-to-noise ratio, an antibody that does not recognize the antigen being studied can be added to the lysate and processed as for a normal immunoprecipitation. Any nonspecific proteins that might contaminate the final immunoprecipitation step are presumably removed with this irrelevant antibody. - [Read Immunoprecipitation: Preclearing the Lysate Protocol]

Category: Immunohistochemistry Protocols

Immunostaining conditions. Includes: Antibody, Host, Source, Cat #, Antigen Retrieval, Block, Diluent, Ab Dilution and 2° antibody. - [Read Immunostaining Conditions]

Category: Signalling Signal Transduction Protocols

Assess T cell activation protocol - measure T cell proliferation upon in vitro stimulation of T cells via antigen or agonistic antibodies to TCR. eBioscience - [Read In Vitro T Cell Activation]

Category: Immunology: Mononuclear Cell Isolation Protocols

Presents two methods for preparing dendritic cells (DCs), a highly specialized type of antigen-presenting cell (APC). The first method involves the isolation of DCs from mouse spleen, resulting in a cell population that is highly enriched in accessory cell and APC function. A support protocol for collagenase digestion of splenocyte suspensions is described to increase the yield of dendritic cells. The second method involves generating large numbers of DCs from mouse bone marrow progenitor cells. - [Read Isolation of Dendritic Cells Protocol]

Category: Signalling Signal Transduction Protocols

Protocol describes how tagged Mos kinase is translated in vitro, immunopurified, and used in a kinase assay. Protocol includes: Translation of Xenopus Mos Kinase; Antibody to Antigen Binding; Protein A Sepharose to Antibody Binding; Kinase Reactions on Immunoprecipitated Material; Polyacrylamide Gel Analysis of Immunoprecipitates. Includes protocol hints. - [Read Kinase Assay Using In Vitro Translated Xenopus Mos Kinase]

Category: Antibody Protocols: Antibody Production Protocols

Protocol for polyclonal antibody production. Very useful for rapid and simple generation of antibodies for western blots, ELISA assays, and immunoprecipitation. Includes: Rabbit Immunization; Initial Preparation; Pre-bleed; Antigen Injection; Monitoring of Titer; Purification of Antibodies. - [Read Polyclonal Antibody Production Protocol]

Category: Fungi Protocols

Protocol for the preparation of solid tissue for Aspergillus galactomannan antigen detection by Platelia (Biorad). Technique was designed for use on human serum. However, it may also be possible to perform this method on solid tissues and organic solutions. Viscous solution and tissue specimens need to be pre-treated to achieve the extraction of the Aspergillus antigen and to get a homogeneous sample in solution. - [Read Preparation of Solid Tissue for Aspergillus Galactomannan Antigen Detection by Platelia Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: T-Cell Isolation Protocols

Protocols utilize T hybridomas to detect expression of peptide-MHC complexes, since these cells provide the most convenient, consistent, and flexible T cell readout systems for these purposes. If desired, antigen-specific T cell clones can be used in lieu of T hybridoma cells, but T cell clones often give poorer responses than T hybridomas to fixed APCs due to fixation-induced loss of costimulator function. - [Read Presenting Exogenous Antigen to T Cells Protocol]

Category: Immunology: B Cell Protocols

Describes procedures for measuring the capacity of purified B cells to undergo proliferation. The method centers on the use of polyclonal stimulating agents (mitogens) because these agents stimulate the majority of B cells and because the alternative (measurement of antigen-induced proliferation) requires the laborious procedures of isolating antigen-specific B cells (which are otherwise present in too low a concentration in whole B cell populations). - [Read Proliferative Assays for B Cell Function Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Sorting Protocols

Protocol for surface antigen staining of cells for fluorescence activated cell sorting. - [Read Protocol for Surface Antigen Staining of Cells for Fluorescence Activated Cell Sorting]