BCH 435 Biochemical Techniques
Experimental studies in the purification and characterization of some enzymes and proteins using biochemical methods.
Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light of the appropriate wavelength is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be inferred through the magnitude of the fluorescence.
Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates enabling much higher sensitivity.
· hCG Pregnancy test (CLICK HERE)
· ELISA Activity (CLICK HERE)
· Animation Quiz (CLICK HERE)
· ELISA Experiment (CLICK HERE)
· Virtual Immunology Lab. (CLICK HERE)
· Lecturers and Demonstrators instructions for conducting a simulated ELISA laboratory in the classroom (CLICK HERE)
· Student instructions for conducting a simulated ELISA laboratory in the classroom (CLICK HERE)
High-performance liquid chromatography (or High pressure liquid chromatography, HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds. HPLC utilizes a column that holds chromatographic packing material (stationary phase), a pump that moves the mobile phase(s) through the column, and a detector that shows the retention times of the molecules. Retention time varies depending on the interactions between the stationary phase, the molecules being analyzed, and the solvent(s) used.
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HPLC is a popular method of analysis because it is easy to learn and use and is not limited by the volatility or stability of the sample compound. The history of the HPLC's evolution started in the 1970's. Modern HPLC has many applications including separation, identification, purification, and quantification of various compounds. It is important for those using HPLC to understand the theory of operation in order to receive the optimum analysis of their compounds. Dear our postgraduate students: For using HPLC, you should have a list of manufacturers, a troubleshooting guide, technical assistance, and a bibliography to help reduce your personal research and referencing time.
Arabic Review Lecture Quiz
Homework: Dear my students, Compare between the types of HPLC according to their principles.
3-Amino Acid Analyzer