Lecture 2 - Centrifugation

Question 1

Describe Immunoffinity Separation

Answer 1

Antibodies specific to surface of cell type is coupled to matrix (e.g. collagen, polysaccharide beads, plastic)
Bound cells are isolated by shaking/degrading matrix

Question 2

Describe Flow Cytometry Analysis: Fluorescence
activated cell sorter

Answer 2

■ Specific cells labelled with Abs paired
to a fluorescent dye
■ Cells pass through laser
beam
■ Cells given a positive or negative
charge depending on whether cell is
fluorescent or not.
■ Cells are deflected by an electric field
into collection tubes according to their
charge

Question 3

Outline the cell fractionation techniques

Answer 3

1. Sonication - Shearing of cell wall/membrane by sound
2. Detergent lysis - Triton x 100
3. French pressure cell - sample placed under high pressure
4. Homogenise - physical disruption with homogenise. Dounce homogeniser - cultured cells
   Potter Elvehjem - soft tissue
   steel blade - firm tissue

Question 4

What is Differential Centrifugation?

Answer 4

Separation based on the size of the
particles. Commonly used in simple pelleting
and in obtaining partially pure
preparation of subcellular organelles
& macromolecules.

Question 5

What happens in differential centrifugation

Answer 5

• Tissues/cells disrupted to release internal insides
  -Crude cell mixture is called the homogenate
  -Larger particles sediment faster than smaller ones
  -Cell homogenate can be centrifuged at a series of progressively higher g forces and times to generate partially purified organelles