L9

Question 1

explain Isoelectric focusing

Answer 1

• Is a electrophoretic technique that separates proteins based on isoelectric point
  
  • Proteins are separated over a pH gradient
    ○ A solution of ampholytes is electrophoresed in a gel to establish the pH gradient
    ○ Protein mixture is applied, electrophoresis (electrolyse that can act as a acid or base in aqeuous solutions) resumed
    Proteins migrate to their respective position

Question 2

explain 2D electrophoresis

Answer 2

• Complex protein mixtures separated in 2 dimensions (charge + size) to create a protein map
  
  • Isoelectric focused in 1st dimension and SDS-PAGE focused in 2nd dimension
  • Gel form isoelectric focusing is laid on top of an SDS gel, then protein seperate by size
    Protein spots stained + analysed

Question 3

explain the process of 2D gel electrophoresis

Answer 3

1. Separation of proteins by pI value
   2. Soaking the gel in SDS solution and fitting it on an SDS PA gel
   3. Separating the proteins by molecular mass with SDS page

Question 4

explain ‘salting in’ and ‘salting out’

Answer 4

• Addition of salt at low ionic strength can INCREASE solubility of a protein by neutralising charges on the surface of the protein + decreasing protein-protein interactions– called salting in
  
  • Addition of salt at high ionic strength decreases solubility because the salt competes for binding to water, leaving less water molecules to itneract with charged protein
  • Protein-protein itneractions icnrease causing aggregation + precipitation– called salting out
  • The salt conc. At which a protein precipitates differs from one protein to another
  • So salting out can be used to fractionate proteins
    Dialysis can be used to remove the salt afterwards if needed

Question 5

explain the Hofmeister series

Answer 5

• Hofmeister identified the ion-specific effects on proteins stability
• The Hofmeister series is the ordering of ions in terms of their ability to affect the solubility of proteins
• Ammonium sulphate is the most common salt used for protein precipitation

REFER TO ONENOTE

Question 6

explain dialysis

Answer 6

• Separation of molecules in solution based on the rate at which they diffuse through semi-permeable membrane
  
  • Dialysis tubing, pores of a defined size
  • The most common use for dialysis is the removal of unwanted small molecules (e.g. Salts, reducing agents, dyes etc.) form protein samples
    ○ Buffer exchange
    ○ Separate large + small native proteins
  • Small molecules + ions equilibrate by diffusion across the membrane
    Th final conc. of molecules is dependent on the volume of the solutions involved

REFER TO ONENOTE

Question 7

what is the CONCENTRATION (final) formula

Answer 7

REFER TO ONENOTE

Question 8

explain centrifuges

Answer 8

• 1st step in msot fractionations
  
  • Widely used procedure
  • Separates the homogenate into different parts/fractions
    Can also separate soluble vs precipitated (insoluble) proteins

REFER TO ONENOTE

Question 9

what is the formula for RCF

Answer 9

REFER TO ONENOTE

Question 10

explain mass spectrometry

Answer 10

• The polypeptide chain is cleaved into smaller fragments (trypsin) and the composition and sequence of each fragment is determined
  • The ions are accelerated by electric charge in the mass analyser and separated by m/z ratio to determine the mass

Question 11

what is the process for Edman degradation

Answer 11

1. Coupling the PITC/Edman reagent to the alpha-amine of the protein chain under basic conditions to form PTC moiety
   2. Cleavage under mild acidic conditions generate a free amino terminus on the protein and an ATZ adducted amino acid
   ATZ-amio acid is extracted and converted to a stable PTH derivative, the pTH residues is analysed by HPLC and compared to PTH amino acids standards

Question 12

what are the 2 types of mass spectrometry

Answer 12

○ Standard mass spectrometry
- Tandem mass spectrometry- has a extra step to fragment further

Question 13

explain X-ray crystallography

Answer 13

• Used to determine the 3D structure of a protein
  
  • A narrow beam of parallel x-rays is directed at a protein crystal
  • A small fraction of the x-rays are scattered in a defined manner, and appear as dots on a detector
  • The position and intensity of the spots gives info on the location of atoms in the crystal
  • Computational modelling allows the determination of the 3D structure of the protein- mainly for tertiary or quaternary structures
    The 3D structure is important in modern day drug discovery

Question 14

what is the process of X-ray crystallography

Answer 14

1. obtaining protein
2. protein purification
3. protein crystallisation
4. data collection
5. structure determination