Unit 1- Lab Techniques Flashcards
What is the difference between linear and log dilutions?
A linear dilution series consists of a range of dilutions that differ by an equal interval. Each solution is made up separately from a stock solution meaning error is not carried on to the next dilution. A log dilution series consists of a range of different dilutions that differ by a constant proportion. Each solution becomes the stock solution for the next dilution meaning any error is carried.
What is colorimetry and what can it be used for?
A colorimeter can be used to measure the concentration of pigment in a solution, the turbidity of liquid or the density of cells in a culture. It is does this by electronically recording the amount of light absorbed in a sample in a small cuvette. A standard curve can be created with an equation from the line of best fit allowing you to determine the concentration of unknown samples.
What are pH buffers and why do we need them?
Buffers are aqueous solutions that show very little variation in their pH despite addition of acids or alkalis. They are used to make sure the pH in experiments remain constant as pH affects most biological systems.
How does centrifugation work?
It separates materials by their density, material is rotated and the resultant g force causes components to separate. The most dense liquid forms a pellet and the liquid above is known as the supernatant.
How does paper and thin-layer chromatography separate proteins?
By their solubility
How does affinity chromatography work?
It used for the separation of one particular protein from a mixture. An antibody or ligand specific for binding with the protein is immobilised in an agarose gel in a column. When the mixture is poured through the column only the protein of interest binds to the antibody or ligand within the column. This protein can then be separated by washing the column with a ph buffer or free ligand.
How does protein electrophoresis work?
This uses current through a buffer to separate the proteins. The tank is set up with a positive and negative terminal. The fragments move towards the electrode with the opposite charge and small fragments will move more quickly as they do not get tangled up in the agarose gel.
What is the iso-electric point of a protein?
The pH at which a protein carries no net electrical charge.
How does iso-electric focusing work?
A pH gradient is set up along a tube of polyacrylamide gel using a mixture of special buffers. Each protein in the sample that is loaded onto the gel will move until it reaches the pH corresponding to its IEP. At this pH, the protein will move no further and form a band which can be visualized after staining.
How are monoclonal antibodies produced?
To produce pure monoclonal antibodies, a single line of B lymphocytes must be grown, each secreting the same specific antibody. B lymphocytes can be harvested from the spleen of a mouse that has been exposed to a specific antigen several weeks earlier. B lymphocytes do not divide in culture. To get around this problem the cells are fused with myeloma (cancer) cells from an immortal cell line, using polyethylene gel (PEG). The cells produced are called hybridomas. Dilution ensures that each hybridoma is placed in its own screening well.
Through the use of selective media, the hybridoma cell line, capable of producing the specific antibody of interest can be identified. Cultures can then grown in fermenters and each immortal hybridoma cell line produces only one type of antibody so it is monoclonal.
The secreted antibody is extracted and purified using centrifugation.
What are potential uses of monoclonal antibodies?
Targeted cancer therapies, detection and diagnosing disease e.g. HIV and meningitis.
How does ELISA work?
ELISA uses monoclonal antibodies that have an enzyme attached. This reporter enzyme catalyses a colour-change reaction that is used to detect and quantify the presence of a specific antigen. If the antigen is present, the antibody binds allowing, the reporter enzyme to produce a coloured product which is visual conformation of the antigen’s presence. If the antigen is not present, the antibody cannot bind and the enzyme is washed away before any reaction can take place.
How does fluorescent labeled protein blotting work?
Used for identifying specific proteins that have been separated by gel electrophoresis. The proteins are blotted from the gel onto a nitrocellulose membrane or nylon filter, recording their final positions on a more convenient material. The filter is then flooded with fluorescent labeled monoclonal antibodies. Because the antibodies are specific to a protein by labelling them you can identify the protein once they have bound. Once the antibodies have bound the excess is washed away. When exposed to light of particular wavelengths the fluorescent labeled antibodies allow the precise location of their specific target proteins to be identified. A fluorescent labeled protein blot allows the location of the target protein to be precisely identified.
What is aseptic technique?
A set of precautions taken to prevent contamination. For example, the use of sterile materials and the appropriate treatment of the source tissue.
How does methylene blue allow you to identify dead and alive cells?
Only dead cells are stained blue whereas live cells are colourless as they are able to actively pump out the stain.
