U1 - Separation Techniques

Question 1

Centrifuge

Answer 1

Separates substances of differing density. More dense settle in the pellet, less dense in the supernatant.

Question 2

Paper and thin layer chromatography

Answer 2

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.

Question 3

Affinity Chromatography

Answer 3

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.

Question 4

Gel electrophoresis

Answer 4

Charged macromolecules move through an electric field applied to a gel matrix.

Question 5

Native gels

Answer 5

Separate proteins by their size, shape, and charge.

Native gels do not denature the molecule.

Question 6

SDS-Page

Answer 6

Separates proteins by size alone.

Gives all molecules an equally negative charge and denatures them

Question 7

Isoelectric Point

Answer 7

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.

Question 8

IEP and Gel Electrophoresis

Answer 8

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.

Question 9

What are stocks of antibodies with the same specificity called?

Answer 9

Monoclonal antibodies

Question 10

What is the technique called that is used to detect and identify proteins?

Answer 10

Immunoassay techniques

Question 11

What is used to detect these antibodies

Answer 11

A chemical ‘label’ which is often a reporter enzyme which produces a colour change.

Question 12

Bright field Microscopy

Answer 12

Used to observe Whole organisms, parts of organisms, thin sections of dissected tissue or individual cells.

Question 13

Fluorescence Microscopy

Answer 13

Uses specific fluorescent labels to bind to and visualise certain molecules or structures within cells or tissues.

Question 14

what is aseptic techniques?

Answer 14

It eliminates unwanted microbial contaminants when culturing micro-organisms or cells.

Question 15

What is involved with aseptic techniques?

Answer 15

Involves the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants

Question 16

What starts a microbial culture?

Answer 16

Can be stared using an inoculum of microbial cells on an ager medium or in a broth with suitable nutrients.

Question 17

Many culture media exist that promote the growth of specific types of cells and microbes.

Answer 17

d

Question 18

What is western blotting ?

Answer 18

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.

Question 19

What are animal cells grown in?

Answer 19

A growth factors from serum

Question 20

What are growth factors?

Answer 20

Are proteins that promote cell growth and proliferation. Growth Factors are essential for the growth of most animal cells.

Question 21

Primary cell lines vs Tumour cell lines

Answer 21

Primary cell lines can divide a limited number of times whereas tumour cell lines can perform unlimited divisions.

Question 22

What does plating out of a solid medium allow?

Answer 22

Allows the number of colony-forming units to be counted and the density of cells in the culture estimated.

Question 23

What is vital staining for?

Answer 23

Vital staining is required to identify and count viable cells