Biotech Final - Lecture 8

Question 1

What are the basic steps of immunoblotting?

Answer 1

1) Coat surface with sample (electroblotting step)
2) Block unoccupied sites with non-specific protein (milk)
3) Incubate with primary antibody against specific antigen
4) Incubate with antibody-enzyme complex that binds primary antibody
5) Add substrate
6) Formation of coloured product indicates presence of specific antigen

Question 2

What is the luminol chemiluminescence?

Answer 2

Addition of luminol and oxidant to blot containing secondary antibody with conjugated HRP. HRP cleaves the substrate to allow for a chemiluminescent reaction to occur.

Question 3

Western blotting is used to?

Answer 3

Identify macromolecular antigens that react with a specific polyclonal or monoclonal antibody.

Question 4

How is Western Blotting accomplished?

Answer 4

1) SDS-PAGE
2) Electroblotting (transfer to PVDF or nitrocellulose)
a) proteins stick to blot in same position
3) Block with inert blocking protein (such as serum albumin or dry milk, to coat unoccupied areas)
4) Incubate with primary antibody
5) Wash
6) Incubate with secondary antibody
7) Chemiluminescence
8) Document using Bio Imaging system

Question 5

The conjugate enzyme catalyzes a chemiluminescent reaction that allows the visualization of as little as _____ of protein.

Answer 5

1 pg