Key Area 1 Flashcards
What are examples of hazards in a lab?
Toxic or corrosive chemicals.
Heat or flammable substances.
Pathogenic organisms.
Mechanical equipment.
What is a risk?
Risk is the likelihood of harm arising from exposure to a hazard.
What does a risk assessment do?
Identifies control measures to minimise the risk.
What are examples of control measures?
Using appropriate handling techniques.
Protective clothing and equipment.
Aseptic technique.
Linear Dilutions
Differ by an equal interval, e.g. 0·1, 0·2, 0·3 …
Log Dilutions
Differ by a constant proportion, e.g. 10-1, 10-2, 10-3 …
What does a standard curve tell us?
Plotting values for known concentrations to produce a line/curve allows the concentration of an unknown to be determined.
What do buffers do?
Allows the pH of a reaction mixture to be kept constant.
Addition of acid or alkali has very small effects on the pH of a buffer.
Use of colorimeter to measure concentration:
Calibration with suitable blank as a baseline.
Use of absorbance to determine conc. of a solution using correct wavelength filter.
Use of colorimeter to measure turbidity:
Use of percentage transmission to determine turbidity.
e.g cells in suspension.
How do solutions separate during centrifugation?
More dense components settle in the pellet (bottom).
Less dense components remain in the supernatant (above pellet).
Paper and thin layer chromatography:
Separate different substances such as amino acids and sugars.
Speed of each solute travels depends on its solubility in the solvent used.
How does affinity chromatography separate proteins?
- Solid matrix/gel column have specific
molecules bound to the matrix/gel. - Soluble target proteins in a mixture with a high
affinity for molecules in matrix/gel attach to
them as the mixture passes them. - Molecules with a weaker affinity are washed
out.
How does gel electrophoresis separate proteins?
2 types:
- Native gels
- SDS-PAGE
Charged macromolecules move through an electric field applied to a gel matrix.
Native Gels
Separate proteins by their shape, size and charge.
Do not denature the molecule.
SDS–PAGE
Separates proteins by size alone.
Gives all the molecules an equally negative charge.
Denatures molecules.
Isoelectric Points (IEPs)
pH at which a soluble protein has no net charge and will precipitate out of solution.
IEPs and buffers:
Only proteins that have an IEP of that specific pH will precipitate.
IEP electrophoresis:
Soluble proteins separated using an electric field and a pH gradient.
Protein stops migrating through the gel at its IEP because it has no net charge.
What are immunoassay techniques used for?
To detect and identify specific proteins using monoclonal antibodies.
Monoclonal antibodies:
Stocks of antibodies with the same specificity.
Chemical labels
Shown when:
- An antibody is specific to the protein antigen.
- A specific antigen detects the presence of
antibodies.
Usually it’s a reporter enzyme producing a colour change.
Other chemical labels include:
- Chemiluminescence
- Fluorescence
Western blotting:
Used after SDS–PAGE electrophoresis.
Separated proteins from the gel are transferred (blotted) onto a solid medium.
How are proteins identified in western blotting?
Using specific antibodies that have reporter enzymes attached.
Bright-field microscopy:
Used to observe 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.
Aseptic technique:
Sterilisation of equipment and culture media by heat or chemical means to eliminate unwanted contaminants when culturing microorganisms or cells.
How is a microbial culture started:
Using microbial cells on an agar medium, or in a broth with suitable nutrients.
There are different culture media for specific types of microbes.
How are animal cells grown?
Grown in a medium containing growth factors from a serum.
What are growth factors?
Proteins that promote cell growth and proliferation.
Essential for the culture of most animal cells.
