antigen Protocols

Category: Immunology: Immunoprecipitation Protocols

Immunoprecopitation method, the protein from the cell or tissue homogenate is precipitated in an appropriate lysis buffer by means of an immune complex which includes the antigen (protein), primary antibody and Protein A-, G-, or L-agarose conjugate or a secondary antibody-agarose conjugate - [Read Protocol Immunoprecipitation]

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

Immunoaffinity purification of antibodies is used to purify antigen-specific antibodies from a preparation of polyclonal antibodies.  Such purification is commonly needed in the production of antipeptide antibodies, where it is used to concentrate the desired antibodies and separate them from those raised against carrier proteins.  It is also used for the more general purpose of removing unwanted, nonspecific binding activity from polyclonal antibody preparations. - [Read Purification of Antibodies on an Antigen Column Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

In an attempt to accurately measure DNA content with simultaneous preservation of cell surface markers, we have utilized gentle ethanol treatment techniques, which permeablize cells with minimal loss of surface antigen expression and antibody-antigen association. For some cell types, the presence of apoptotic cells based on reduced DNA content can also be detected. One such technique employs the addition of ethanol to cells previously resuspended in high concentrations of fetal bovine serum... - [Read Simultaneous Analysis of DNA Content and Surface Immunophenotype Protocol]

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

INDO-1 loading and sample staining protocol for simultaneous measurement of intracellular CA2+ and cell surface antigen expression. Includes: Preparation of INDO-1; Preparation of Ionomycin. - [Read Simultaneous Measurement of Intracellular CA2+ and Cell Surface Antigen Expression Protocol]

Category: Transfection Protocols: Transient Transfection Protocols

Transient transfection into 293T cells is a convenient way to overexpress and obtain both cellular and extracellular (secreted or membrane) proteins. 293 is a human renal epithelial cell line which is transformed by adenovirus E1A gene product. 293T is a derivative which also express SV40 large T antigen, allowing episomal replication of plasmids containing the SV40 origin and early promoter region. They (both) have the unusual property of being highly transfectable. - [Read Transient Transfection Into 293T Cells Protocol]

Category: Immunohistochemistry Protocols: Antigen Retrieval Protocols

Several methods have been developed to "retrieve" antigens that have been masked by fixation. The principle behind using the microwave oven method described here is to use extended periods of heat to break some of the subcellular structures that block antibody access. Be aware that any of the antigen retrieval methods should be avoided wherever possible, because they may introduce artifactual false-positive staining. - [Read Unmasking Hidden Epitopes Using the Microwave Oven Protocol]

Category: Immunohistochemistry Protocols: Antibody Staining Protocols

Protocol for VEGF antibody staining. Includes: Primary Antibody; Antigen Retrieval; Detection Methods; Chromogen Substrate; Counterstain. - [Read VEGF Antibody Staining Protocol]

Category: Western Blot Protocols: Antigen Detection Methods Protocols

Use of the chemiluminescence-producing alkaline phosphatase substrate 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1.1(3,7)]decan]-4-yl)phenyl phosphate (AMPPD, also known as adamantyl-1,2-dioxetane phosphate), or its dioxetane relatives provides a substantial increase in sensitivity over colorimetric substrates and radiochemical methods currently used for the detection of antigen-antibody complexes immobilized on nylon or PVDF membranes. - [Read Western Analysis Using the Chemiluminescent Alkaline Phosphatase Substrate CSPD Protocol]