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Radiation Safety

Immunohistochemistry

By National Diagnostics | September 20, 2011 | Comments Off on Immunohistochemistry

Immunohistochemistry is the application of antibody/antigen interactions to provide information about biological systems. The body’s response to the introduction of a foreign agent, known as the immune response, results in the production of antibodies which…

Aldehyde Fixatives

By National Diagnostics | September 20, 2011 | Comments Off on Aldehyde Fixatives

Formaldehyde and glutaraldehyde are the most commonly used aldehyde fixatives. They work by forming cross-links both within and between proteins, particularly between lysine residues. Damage to the tertiary structure of the proteins occurs on a…

Overview of Fixation

By National Diagnostics | September 20, 2011 | Comments Off on Overview of Fixation

To maintain the tissue in as lifelike a state as possible, tissue for analysis is usually placed directly into a fixative solution upon removal from the body. Fixation is normally carried out as soon as…

Overview of Western Blotting

By National Diagnostics | September 19, 2011 | Comments Off on Overview of Western Blotting

Proteins can also be detected immunologically following electrophoresis, a technique known as Western blotting. This method relies on the fact that most epitopes (sites recognized by antibodies, generally comprising several amino acids) are still recognizable…

Enzyme Linked Immunosorbent Assay (ELISA)

By National Diagnostics | September 19, 2011 | Comments Off on Enzyme Linked Immunosorbent Assay (ELISA)

One of the most straightforward applications of immunological detection is the ELISA or enzyme-linked immunosorbent assay. In the simplest system, the bound antigen is probed with antibodies that carry covalently attached enzyme molecules. Antibody binding…

Immunostaining with Horseradish Peroxidase

By National Diagnostics | September 19, 2011 | Comments Off on Immunostaining with Horseradish Peroxidase

Chromogenic and luminometric substances are also available for horseradish peroxidase (HRP). The “classic” chromogen used with HRP is diaminobenzidine (DAB). In the presence of H2O2, HRP will oxidize DAB, creating a water insoluble brown precipitate.…

Immunostaining with Alkaline Phosphatase

By National Diagnostics | September 19, 2011 | Comments Off on Immunostaining with Alkaline Phosphatase

Alkaline phosphatase catalyzes the removal of a phosphate group from its substrate. A variety of synthetic substrates have been constructed which, on phosphate hydrolysis, liberate chromogens or luminescent compounds. A commonly used chromogenic substrate is…

Mechanism of Immunostaining

By National Diagnostics | September 19, 2011 | Comments Off on Mechanism of Immunostaining

The highly specific binding interaction between antibodies and their unique antigens has been exploited to create sensitive and specific detection systems for proteins. An antibody can be raised and/or purified “against” (i.e. binding to) a…

Staining Proteins Immobilized on Membranes

By National Diagnostics | September 19, 2011 | Comments Off on Staining Proteins Immobilized on Membranes

Immunological detection of proteins requires that proteins be transferred and immobilized onto a membrane support after electrophoresis (see Western Blotting). Staining of the immobilized proteins establishes transfer efficiency, and allows the operator to mark the membrane…

Guide Strip Technique

By National Diagnostics | September 19, 2011 | Comments Off on Guide Strip Technique

In certain instances, the effects of staining a protein may interfere with subsequent analysis. Examples are Coomassie staining when enzymatic activity is required, or silver staining prior to amino acid analysis when covalent modification of…