L2 - Protein Analysis

Question 1

What is proteomics?

Answer 1

Analysis of complete protein content in a living system.

Secretory fluids have a mix of proteins.

Question 2

Protein purification protocol

Answer 2

1. Tissue homogenisation by sonication, blending or pestle and mortar
2. Separation of released material by centrifugation
3. Chromatography - size exclusion, affinity and ion exchange.
4. Confirmation of protein purity by electrophoresis
5. Confirmation of protein identity by immunoblotting and mass spec

Question 3

Differential centrifugation protocol

Answer 3

1. Cell homogenate mixed at low speed
2. Supernatant mixed at medium speed
3. Supernatant mixed at high speed
4. Supernatant mixed at very high speed to get pure cytosol

Question 4

Density based ultracentrifugation protocol

Answer 4

Sucrose gradients used to separate components

1. Velocity sedimentation
2. Centrifugation
3. Fractionation

Question 5

Gel filtration chromatography protocol

Answer 5

1. Solvent flow over controlled size porous beads
2. Small proteins enter bead and are slowed
3. Large proteins are excluded so are not slowed
4. Fractions then collected

Question 6

Affinity chromatography protocol

Answer 6

Purifies specific DNA binding protein

1. Solvent flow over beads with covalently attached substrate
2. Everything but protein with specific interaction for ligand is removed in the flow through
3. Bound protein is eluted using a competing ligand

Question 7

Ion exchange chromatography protocol

Answer 7

1. Solvent flow over positively charged beads
2. Proteins with a - charge will be attracted and slowed 3. Increasing salt concentrations used to compete for ionic interaction
3. Proteins eluted from the column
4. Fractions then collected

Question 8

SDS page protocol

Answer 8

Protein migration is proportional to molecular mass

1. Proteins boiled for 2 minutes in solution containing mercaptoethanol and SDS to denature them
2. Gives them uniform - charge

Question 9

2D gel electrophoresis for complex samples uses both?

Answer 9

1D - isoelectric focussing separates by charge
- relies on stable pH gradient
- At the isoelectric point the protein has no net charge and therefore no longer migrates in electric field
2D - SDS PAGE separates by size (initial separation)

Question 10

Immunoblotting protocol

Answer 10

Uses 2 antibodies to identify the protein
For 2D electrophoresis
1. All proteins electrophoretically transferred to membrane
2. Membrane incubated with specific antibody against component of mixture
3. Western blot developed using chemicals that are converted to light
4. Reaction revealed as coloured spots or using camera

Question 11

Mass spec protocol

Answer 11

1. Peptide ionisation and measurement of their mass
   
   • Trypsin breaks up peptide chains
2. Ions separated according to their mas-charge ratio
3. Detected in proportion to their abundance

Question 12

Determination of protein phosphorylation by mass spec

Answer 12

3 amino acids can be phosphorylated because they have OH groups

• Serine
• Threonine
• Tyrosine

Question 13

Cation exchange in ion exchange chromatography

Answer 13

CM carboxy-methyl

Question 14

Anion exchange in ion exchange chromatography

Answer 14

DEAE diethylaminoethyl