Cells and Proteins Flashcards
What things in labs can present a hazard?
Substances, organisms and equipment.
Hazards in the lab include?
- Toxic or corrosive chemicals
- Heat or flammable substances
- Pathogenic organisms
- Mechanical equipment.
What is risk?
Likelihood of harm arising from exposure to a hazard.
What is a risk assessment?
Involves identifying control measures to minimise the risk.
Control measures include using…
- Appropriate handling techniques
- Protective clothing and equipment
- Aseptic technique
Linear dilution series?
Differ by an equal interval e.g. 0.1, 0.2, 0.3 and so on
Log dilution series?
Differ by a constant proportion, e.g. 10^-1, 10^-2, 10^-3
Production of a standard curve to determine an unknown?
Plotting measured values for known connections to produce a line or curve allows the concentration of an unknown to be determined from the standards curve.
Use of buffers to control pH?
Additional of acid or alkali has a very small effect on the pH of a buffer, allowing the pH of a reaction mixture to be kept constant.
Method and uses of a colorimeter to quantify concentration and turbidity?
Calibration with appropriate blank as a baseline; use of absorbance to determine concentration of a coloured solution using suitable wavelength filters; use of % transmission to determine turbidity, such as cells in suspension.
Use of a centrifuge to separate substances of differing density - what is supernatant and pellet?
More dense components settle in the pellet and less dense components remain in the supernatant.
Paper and thin layer chromatography use?
Used to seperate different substances such as amino acids and sugars.
Paper and thin layer chromatography - how does it work?
The speed that each solute travels along the chromatography depends on its differing solubility in the solvent used.
Principe of affinity chromatography and its use in separating proteins?
A solid gel matrix of gel compound is created with specific molecules bound to the matrix or gel. Soluble target proteins in a mixture, with high affinity for these molecules become attached to them as the mixture passes down the column.
Non target proteins with a weaker affinity are washed out.
Principle of gel electrophoresis and its use in separating proteins and nucleic acids?
Charged macromolecules move through an electric field applied to a gel matrix.
Native gels?
Do not denature the molecule so that separation is by shape, size and charge.
SDS-PAGE?
Gives all molecules an equally negative charge and denatures them, separating proteins by size alone.
What is IEP?
The pH at which a soluble protein has no net charge and will precipitate out of solution.
Proteins can be seperated from a mixture using their IEPs - how?
If the solution if buffered to a specific pH, only the proteins(s) that have an IEP of that pH will precipitate.
How can proteins be seperated using their IEPs in electrophoresis?
Soluble proteins can be separated using an electric field and a pH gradient. A protein stops migrating through the gel at its IEP in the pH gradient because it has no net charge.
What is immunoassay techniques used for?
To detect and identify specific proteins.
What does immunoassay use?
Monoclonal antibodies.
What are monoclonal antibodies?
Stocks of antibodues with the same specificity.
An antibody specific to the protein antigen is linked to a chemical ‘label’ - what is this label often?
A reporter enzyme producing a colour change, but chemiluminescence, fluorescence and other reporters can also be used.
