Lab Techniques Flashcards
Name 4 hazards within a lab
Toxic/corrosive substances
Heat/flammable substances
Pathogenic organisms
Mechanical equipment
What are appropriate control measures for a lab
using appropriate handling techniques
protective clothing
aseptic technique
What does a centrifuge separate substances based on
The substance’s density
Where do the more and less dense components settle in a centrifuge
The more dense end up in the pellet
The less dense end up in the supernatant
What can be used to separate different substances such as amino acids and sugars
paper/thin-layer chromatography
What determines the speed that the solute runs along the chromatogram
The solubility of solute in solvent
How does affinity chromatography work
A solid matrix/gel column is created with specific molecules bound to the matrix or gel
Soluble, target proteins in a mixture with a high affinity for these molecules, becomes attached to them as the mixture passes down the column. Other non-target molecules with a weaker affinity are washed out.
How does gel electrophoresis work
Charged macromolecules move through an electric field applied to the gel matrix
What do native gels separate proteins based on and how is this achieved?
By shape, size and charge
This is because native gels do not denature the molecule
What does SDS-PAGE separate proteins based on and how is this acheived
Size alone since SDS-page gives all the molecules an equally negative charge and denatures them, separating proteins by size alone
What is the IEP
The pH at which a soluble protein has no net charge and will precipitate out of the solution
How can IEPs be used in electrophoresis
Soluble proteins can be separated using an electric field and a pH gradient. A protein stops migrating through a gel at its IEP in the pH gradient because it has no net charge.
What is the name of stock antibodies used in immunoassay
Monoclonal antibodies
What does immunoassay do
It is used to detect and identify specific proteins
Describe the process of immunoassay detection
An antibody specific to the protein antigen is linked to a chemical label. Upon binding to the protein antigen a colour change/chemiluminescence/ fluorescence is produced
When is western blotting used
after SDS-PAGE electrophoresis
Describe western blotting
Separated proteins from the gel are blotted on a solid medium. Proteins can be identified through immunoassay
What is bright-field microscopy used for
To observe whole organisms, parts of organisms, thin sections of dissected tissue or individual cells
How does fluorescence microscopy work
It used specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues
What does aseptic technique eliminate
Unwanted microbial contaminants when culturing micro-organisms or cells
What does aseptic technique involve
The sterilisation equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants
What are animal cells grown in
Medium containing growth factors from serum
What are growth factors
Proteins that promote cell growth and proliferation
How many times can primary cell lines divide
Limited times
How many times can tumour cell lines divide
Unlimited number of times
How is the number of colony-forming units counted and density of cells in culture estimated
Plating out of liquid microbial culture on solid media
What is required for a suitable colony count
Serial dilution
What apparatus is used to estimate cell numbers
Haemocytometer
What is used to identify and count viable cells
Vital staining
