Laboratory Techniques Flashcards
What is a linear dilution?
Dilutions that differ by an equal interval, e.g. 0.1M, 0.2M, 0.3M etc
What is a log dilution?
A dilution that differs by a constant proportion, e.g. x10^1, x10^2, x10^3 etc
What is a standard curve used for?
Determining the concentration of an unknown solution.
What are used to control the pH of solutions?
Buffers
Explain what a colorimeter measures and what it is used for.
A colorimeter can be used to quantify the concentration and turbidity of a solution. The colorimeter is calibrated using an appropriate blank as a baseline. The measurement of absorbance is used to determine the concentration of a coloured solution using suitable wavelength filters.
Explain what centrifugation is.
Centrifugation is a technique used to separate substances of differing density. More dense components settle in a pellet and less-dense components remain in the supernaut.
Explain what is meant by paper and thin layer chromatography.
They can be used for separating different substances such as amino acids and sugars. The speed that each solute travels along the chromatogram depends on its solubility in the solvent used.
What is affinity chromatography and how does it work?
Affinity chromatography is a separation technique in which soluble target proteins with a high affinity in a mixture become attached to specific molecules as they pass down a column. Non-target molecules with a weaker affinity are washed out. The target protein can then be washed out separately and collected.
Explain what gel electrophoresis is.
Gel electrophoresis is a technique that can be used to separate proteins and nucleic acid. In gel electrophoresis, charged macromolecules move through an electric field applied to a gel matrix.
State the two types of gel electrophoresis and explain the differences between them.
Native and SDS-page. Native separates proteins and nucleic acid by their size, shape and charge and doesn’t denature the molecule whereas SDS-page gives all the molecules an equally negative charge and denatures them, separating proteins by size alone.
What is an isoelectric point?
The IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution.
What happens to a protein when it reaches its IEP?
It precipitates out of the mixture.
What are immunoassay techniques used for?
To detect and identify specific proteins.
What are monoclonal antibodies?
Stocks of antibodies with the same specificity.
Explain the process of immunoassay techniques.
An antibody specific to the protein antigen is linked to a chemical ‘label’. The label is often a reporter enzyme producing a colour change, but chemiluminescene, fluorescene radioactivity and other reporters can be used. In some cases, the assay uses a specific antigen to detect the presence of antibodies.
