Lab Techniques 2

• "Assay" means a procedure used for detecting the presence and quantity of a substance.
• Immunoassays are tests based on the specific binding that occurs between an antibody and the antigen that it specifically recognises.
• Immunoassay techniques are used to detect and identify specific proteins.
• When immunoassays are used to test for the presence of an antibody, the test contains the antigen as part of the detection system.
• If the antibody being tested for is present, it will bind to the antigen in the test system and will be detected- a positive result.
• When used to test for the presence of antigens, the test contains antibodies against the antigen.
• The reaction of the antigen that is present in the sample to the specific antibody is compared with reactions of known concentrations and the concentration of antigen is calculated.
• These techniques use stocks of antibodies with the same specificity known as monoclonal antibodies.
• Monoclonal antibodies are antibodies derived from a single cell line.
• To see if the antigen has bound, another antibody specific to the antigen is added, and is linked to a chemical label.
• The label is often a reporter enzyme that will produce a colour change, but chemiluminescence, fluorescence and other reporters can be used.
• Western blotting is a technique used after SDS-Page Gel Electrophoresis
• After SDS-page, the separated proteins from the gel are transferred onto a solid medium.
• In western blotting, proteins can be identified using specific antibodies that have reporter enzymes attached.
• ELISA tags the antibodies with certain enzymes. If the target antigen is present, the enzyme causes a reaction which produces a colour change.
• Several washes are repeated between each ELISA step to remove unbound materials.
• During ELISA, it is essential that excess liquid is removed in order to prevent the dilution of the solutions being added in the next stage.
• Bright Field Microscopy uses a bright light source, and the light passing through the specimen gives an image.
• Bright Field Microscopy allows you to look at whole organisms, parts of organisms, thin sections of dissected tissue or individual cells.
• Fluorescence Microscopy uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues.
• A fluorescent molecule is one which absorbs a specific wavelength of light then emits a different wavelength.
• Aseptic technique eliminates unwanted microbial contaminants when culturing micro-organisms or cells.
• Aseptic techniques are used when handling cells to maintain sterile conditions and minimise the risk of contamination.
• Examples of aseptic techniques include disinfection of work surfaces, washing of hands before and after carrying out practical work, and use of personal protective equipment such as a lab coat and safety glasses.
• Cell culture is the ability to grow cells in an artificial laboratory environment.
• Animal cells are grown in medium containing growth factors from serum.
• Growth factors are proteins that promote cell growth and proliferation.
• A cell line is a genetically uniform cell culture developed from a single cell.
• Primary cell lines are cells which are placed into cultures and sub-cultured into fresh medium every few weeks.
• After the first subculture, primary cell lines can only divide a limited number of times before the cells die.
• To prevent cell death, primary cells must be subcultured into fresh media.
• Cell lines derived from primary cultures have a limited lifespan.
• Tumour cell lines are immortal. They can grow and divide indefinitely in culture, even after repeated subcultures
• A haemocytometer is an instrument used to allow the number of cells in a sample to be counted when viewed under a microscope.
• The haemocytometer fluid should be in a homogenous suspension. Cells that stick together in clumps are difficult to count and are not evenly distributed.
• If the concentration of cells in a haemocytometer is too high, then the cells overlap, and are difficult to count
• If the concentration of cells in a haemocytometer is too low, there is a higher risk of statistical error, and it is necessary to count more squares.
• A cell count is used when it is necessary to know the number of cells in a sample.
• A haemocytometer can be used to provide an estimate of both the total and viable cell number.
• The viable cell count is the number of cells that are viable (alive and capable of growth) in a given area or volume