Molecular Diagnosis 14i

Question 1

What is the difference between DNA gel electrophoresis and protein gel electrophoresis, other than the obvious?

Answer 1

The protein gel electrophoresis uses acrinomide gel instead of agarose

Question 2

How is SDS-PAGE prepared?

Answer 2

β-ME - rotten egg smell, reducing agent, breaks disulphide bonds so proteins don’t stick together
SDS - detergent, adds negative charge to molecules, breaks down the secondary structure to form a linear polypeptide chain
This ensures the proteins have the same charge and shape so only weight affects it

Question 3

What is the difference between SDS-PAGE and 2D-PAGE?

Answer 3

SDS - separates proteins on basis of size

2D - separates complex mixtures of proteins on basis of isoelectric point (the weird one)

Question 4

What is an epitope?

Answer 4

A few amino acids on a protein which can be recognised by an antibody

Question 5

What are the two types of antibodies?

Answer 5

Polyclonal - produced by many β- lymphocytes, multiple different antibodies, specific to one antigen, multiple epitopes
Monoclonal - produced from 1 β-lymphocytes 1 identical antibody, specific to 1 antigen, 1 epitope (more important clinically)

Question 6

How do you measure the rate of an enzyme catalysed reaction?

Answer 6

Using enzyme assays
Measure the product by spectrophotometry (continuous), chemoluminescence (continuous), radioactivity (discontinuous) or chromatography (discontinuous)

Question 7

Why might you measure enzymes in a patient?

Answer 7

For metabolic disorders (in tissues), and diagnosis of disease (serum enzymes)

Question 8

What are some clinically important serum enzymes and what do they check for?

Answer 8

AST - liver damage/disease
ALT - same as above
Amylase or lipase - pancreatitis
γ-glutamyl transferase - liver damage, increased by alcohol
Alkaline phosphatase - bone disorders
CK - myocardial infarction, use antibodies to detect presence of enzymes (eg lactate dehydrogenase)