Molecular Techniques - Proteins

Question 1

Which techniques can be used for protein analysis?

Answer 1

1. Protein electrophoresis
2. Immunoassays
3. Enzyme assays

Question 2

What is protein electrophoresis and what are its requirements?

Answer 2

• Proteins are charged molecules - will move towards the anode or cathode if placed in an electric field. Can be separated on the basis of size, charge or shape.
• Requirements:
  ~ gel - matrix that allows separation of the protein sample
  ~ buffer - maintains charge on the protein samples
  ~ power supply - generates charge difference across the gel
  ~ stain/detection - identify the presence of the separated proteins

Question 3

Which type of protein electrophoresis is used to separate proteins on the basis of size?

Answer 3

• SDS-PAGE

- SDS (sodium dodecyl sulphate) breaks down the 3D structure, forming a linear polypeptide.

Question 4

Which type of protein electrophoresis is used to separate proteins on the basis of charge?

Answer 4

• IEF (isoelectric focusing)

i) A stable pH gradient is established in the gel after application of an electric field.
ii) Protein solution is added and electric field is reapplied.
iii) Proteins migrate until they reach a pH equal to their pI.

Question 5

Which type of protein electrophoresis is used to separate complex mixtures of proteins?

Answer 5

• 2D-PAGE

- Separates first on the basis of pI then on basis of size.

Question 6

What are immunoassays?

Answer 6

• Monoclonal antibodies bind to specific protein targets (antigens) by recognising a few amino acids on the antigen (epitope).

Question 7

Describe 3 types of immunoassay.

Answer 7

1. Western blotting - protein detection
   - nitrocellulose replica of gel electrophoretogram
   - binding of primary antibody
   - binding of enzyme-linked secondary antibody
   - immunoblot
2. ELISA (enzyme-linked immunosorbent assay) - measures protein concentration in solution
   - antigen coated well
   - specific antibody binds to antigen
   - enzyme-linked antibody binds to specific antibody
   - substrate is added and converted by enzyme into coloured product; the rate of colour formation is proportional to the amount of specific antibody
3. Radioimmunoassay
   - Works in same way but uses radiolabelled primary antibody.

Question 8

Which type of assays can be used to measure product formation in enzyme assays?

Answer 8

• Continuous assays:
  ~ spectrophotometry
  ~ chemoluminescence
• Discontinuous assays:
  ~ radioactivity
  ~ chromatography

Question 9

When are enzyme assays used?

Answer 9

• To measure enzyme activity.
• Important in:
  1. Metabolic disorders (in tissues)
  2. Disease diagnosis (serum enzymes)

Question 10

Give an example of the use of enzyme assays in disease diagnosis.

Answer 10

Enzyme elevation after acute MI. Can measure:

• creatine kinase isoenzymes (elevated after MI)
• cardiac troponin I by ELISA - now gold standard for diagnosis