Unit 1: Lab Techniques Flashcards
(17 cards)
How is a centrifuge used to separate substances in solution?
A centrifuge separates substances of different density with more dense components settling in the pellet and less dense components remaining in the supernatant.
Describe how affinity chromatography is used
A solid matrix or gel is created with specific molecules bound to the matrix or gel. The soluble target proteins that are in the mixture with a high affinity for these molecules, become attached while other non-target molecules are washed out.
Describe the process of gel electrophoresis
Charged macromolecules move through an electric field applied to a gel matrix.
How do native gels separate proteins?
Native gels do not denature the molecule so that separation is by shape, size and charge.
How does SDS PAGE separate proteins?
SDS–PAGE gives all the molecules an equally negative charge and denatures them, separating proteins by size alone.
How else can proteins be separated?
By their isoelectric point (IEP)
What is the isoelectric point of a protein?
IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution. Proteins can also be separated using their
IEPs in electrophoresis
What is western blotting and when is it used?
Western blotting is a technique, used after SDS–PAGE electrophoresis
The separated proteins from the gel are transferred (blotted) onto a solid medium
What is used to detect proteins?
Antibodies
How are specific proteins identified?
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
chemiluminescence, fluorescence and other reporters can be used.
What are aseptic techniques used for?
Aseptic technique eliminates unwanted microbial contaminants when culturing micro-organisms or cells
What are aseptic techniques?
Aseptic technique involves the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of
microbial contaminants.
What are examples of control measures?
Appropriate handling techniques, protective clothing and equipment and aseptic techniques
How much can primary cell lines divide?
A limited number of times
How much can tumour cells divide?
An unlimited number of times
What is used to estimate cell count?
A haemocytometer
What is used to identify and count viable cells?
Vital staining
