Applications

ELISA

What is an ELISA? The well-known term ELISA is an abbreviation of Enzyme-Linked Immunosorbent Assay, yet although ELISA readouts have traditionally relied on the use of an antibody which has been linked to an enzyme, the term is now broadly used to describe any plate-based immunoassay in which a molecule has been adsorbed on to…

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Flow Cytometry

Flow cytometry is a technique which is used to analyze the characteristics of cells as they flow singly past one or more beams of focused light, provided by lasers. When the cells pass through the laser beam they scatter the light, which is detected as forward scatter (correlating to cell size) and side scatter (correlating…

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Fluorescence Polarisation

Fluorescence polarization indirectly measures the speed of rotation of molecules in a solution. A motionless fluorescent molecule that is excited with plane-polarized light will, after a short lag (the ‘fluorescence lifetime’), emit light polarized in the same plane. If rotation of the excited molecule occurs during the fluorescence lifetime (usually a few nanoseconds) the emitted…

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FRET

Fluorescence Resonance Energy Transfer (FRET) assays Fluorescence occurs when a fluorophore moves to an excited state following the absorption of light at a specific wavelength, and subsequently produces a transient light emission at a higher wavelength as it returns to its ground state. The emitted light can be detected with a specialized reader. Fluorescence Resonance…

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High Throughput Screening

Evolution of the drug discovery process The discovery of penicillin by Sir Alexander Fleming in 1928 is widely considered to be one of mankind’s greatest medical advances. This antibiotic, produced by the fungal genus Penicillium, is effective against a wide range of Gram-positive bacteria, including the causative agents of pneumonia and septicemia. The outbreak of…

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Immuno-PCR

Introduction Immuno-PCR is an extremely powerful method of immunodetection which combines the specificity of an ELISA with the signal amplification of PCR. Since an ELISA requires the use of antibodies, it is adaptable to any protein, however its sensitivity is not adequate for the detection of analytes of low abundance. While real time PCR provides…

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Immunocytochemistry

Immunocytochemistry Immunocytochemistry (ICC) is a technique for the visualization of proteins and peptides in cells using biomolecules capable of binding the protein of interest. Usually the biomolecule is an antibody that is linked to a reporter, e.g. a fluorophore, fluorescent dye, or enzyme. The reporter will give rise to a signal, e.g. fluorescence or color…

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Immunofluorescence

What is immunofluorescence? Immunofluorescence is the process by which an antigen is detected with an antibody and is then visualized using a fluorophore. The fluorescent readout is generated by using a light source to excite the fluorophore, which then produces a transient light emission as it returns to its ground state. The emitted light has…

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Immunohistochemistry

Principles of Immunohistochemistry (IHC) Immunohistochemistry (IHC) is the process of using antibodies to detect proteins (antigens) in cells within a tissue section (for instance liver, pancreas or the heart). This tool is used to localize specific antigens in tissue sections with labeled antibodies based on antigen-antibody interactions. The immune reactive products can be visualized by…

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Immunoprecipitation

What is immunoprecipitation? Immunoprecipitation (IP) is a method of isolating a specific protein from a complex mixture such as a cell lysate, tissue homogenate or blood sample. The protein is captured by an antibody, and the antibody-antigen complex is pulled out of the sample by virtue of antibody attachment to a bead. When performing an…

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Lateral Flow Immunoassay

Lateral flow immunoassays, also known as immunochromatographic assays or strip tests, are unidirectional assays which are used to quickly and easily establish whether a target analyte is present in a test sample. The advantages of the lateral flow immunoassay system are well-known: High sensitivity and specificity Requires only a low sample volume Applicable to a…

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The Dot Blot

A simple yet effective detection method Dot blots are very similar to Western blots in that they involve the use of antibodies to identify a protein that has been bound to a membrane. They do not however require electrophoretic protein separation on a gel; the test sample is simply spotted on to the membrane and…

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Time-Resolved Fluorescence

Fluorometric detection typically relies on the use of an antibody which has been labeled with a fluorophore. Once the antibody has bound to its target, a light source is used to excite the fluorophore, which then produces a transient light emission as it returns to its ground state. The light is emitted at a higher…

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Western Blot

The aim of Western blotting is to identify specific proteins within a complex mixture. The Western blot technique requires samples to be resolved on the basis of size through Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), following which they are transferred to, and immobilized on, a membrane prior to antibody-based detection. Western Blot Indirect and…

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