Analysis of biological macromolecules

Question 1

What is the structure and origin of antibodies?

Answer 1

Constant and variable regions connected by a disulphide bridge hinge. The antigen binding site is very specific.
Made by B lymphocytes (each resting B lymphocyte has an antibody on its surface acting as a receptor) when the antigen binds to the receptor it stimulates the division and proliferation of plasma cells which secrete antibodies.

Question 2

What is the process of enzyme linked antibodies?

Answer 2

Using antibodies to detect proteins

1) make antibody
2) add antibody to a solution containing a known enzyme that will provide a colour change
3) wash
4) add secondary antibody
5) wash
6) add detection reagent

Question 3

Why are secondary antibodies used in the detection of proteins?

Answer 3

Amplification - lots of secondary antibodies can bind to 1 primary antibody so it produces a clearer colour change.

Question 4

What is immunoblotting?

Answer 4

Used to separate proteins in terms of size.
Electrophoresis is used and the smallest proteins move fastest through the gel.
transferred into nitrocellulose and antibodies and detection reagent is added.

Question 5

What is ELISA?

Answer 5

Enzyme-linked immunosorbent assay
Used to determine the amount of protein
Cells of protein extracts are added to the bottom of plastic wells.
Primary and secondary antibodies are added along with detection reagent.
The amount of product is proportional to the amount of antibodies.