U1 - Separation Techniques Flashcards
Centrifuge
Separates substances of differing density. More dense settle in the pellet, less dense in the supernatant.
Paper and thin layer chromatography
Separates substances such as amino acids and sugars.
The speed that each solute travels along the chromatogram depends on it differing solubility in the solvent used.
Affinity Chromatography
separates protein
A solid matrix or 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, become attached to them as the mixture passes down the column. Other non-target molecules with a weaker affinity are washed out.
Gel electrophoresis
Charged macromolecules move through an electric field applied to a gel matrix.
Native gels
Separate proteins by their size, shape, and charge.
Native gels do not denature the molecule.
SDS-Page
Separates proteins by size alone.
Gives all molecules an equally negative charge and denatures them
Isoelectric Point
Separates proteins
IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution.
If the solution is buffered to a specific pH, only the proteins that have an IEP of the pH with precipitate.
IEP and Gel Electrophoresis
Separates proteins
Soluble proteins can be separeted using an electric field and pH gradient. A protein stops migrating through the gel at its IEP in the pH gradient because it has no net charge.
What are stocks of antibodies with the same specificity called?
Monoclonal antibodies
What is the technique called that is used to detect and identify proteins?
Immunoassay techniques
What is used to detect these antibodies
A chemical ‘label’ which is often a reporter enzyme which produces a colour change.
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.
what is aseptic techniques?
It eliminates unwanted microbial contaminants when culturing micro-organisms or cells.
What is involved with aseptic techniques?
Involves the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants
What starts a microbial culture?
Can be stared using an inoculum of microbial cells on an ager medium or in a broth with suitable nutrients.
Many culture media exist that promote the growth of specific types of cells and microbes.
d
What is western blotting ?
Is a technique used after SDS-Page. the separated proteins are transferred onto a solid medium and can be identified by specific antibodies that have reporter enzymes.
What are animal cells grown in?
A growth factors from serum
What are growth factors?
Are proteins that promote cell growth and proliferation. Growth Factors are essential for the growth of most animal cells.
Primary cell lines vs Tumour cell lines
Primary cell lines can divide a limited number of times whereas tumour cell lines can perform unlimited divisions.
What does plating out of a solid medium allow?
Allows the number of colony-forming units to be counted and the density of cells in the culture estimated.
What is vital staining for?
Vital staining is required to identify and count viable cells
