Prac 4

Question 1

Separation of proteins (SDS-PAGE)

Answer 1

Polyacrylamide gel (PAG) – acrylamide and bis-acrylamide, defined pore size
SDS (sodium dodecyl sulphate) – equalization of charge and structure
β-mercaptoethanol – reduction of disulphide bridges
Marker – proteins with known molecular weight

Question 2

Electrophoresis (E)

Answer 2

Separation of charged molecules in an electric field
Migration depends on
• the charge, size and shape of the molecules
• the strength of the field
• the ionic strength, viscosity and temperature of the medium

Question 3

Western blot

Answer 3

Detection of proteins
1. Non-specific 
Staining (Coomassie Brillant Blue,Amido Black)
2. Specific 
Enzyme immunoassay 
Chemiluminescence 
Fluorescence 
Radioisotopes

Question 4

Fluorescence Activated Cell Sorting (FACS)

Answer 4

Automated immunofluorescence technique More features of cells can be examined simultaneously

1. Liquid system-Transfer of cells
2. Optical system - Laser beam Detectors
3. Electronical system - Computer

FSC (Forward Scattered Light) Cell size
SSC (Side Scattered Light) Structure and granulatedness of cells

Question 5

Rapid diagnostic tests

Answer 5

• Can be made at the site of examination
• Few minutes – one hour
• Ab or Atg
• Sensitivity

Factors influencing immunogenicity have to be taken into consideration!