Lecture 13 - Proteomics

Question 1

What is the structure of an antibody?

Answer 1

Has 4 chains –> 2 light, 2 heavy
has 3 fragments, 2 Fab (antigen-binding), 2 Fc (crystallizable)
chains held together by noncovalent interactions and disulfide bonds

Question 2

What do antibodies bind to?

Answer 2

specific site (epitope) on an antigen

Question 3

What is the difference between polyclonal and monoclonal antibodies?

Answer 3

pcAbs –> from different B cells, distinct antibodies that recognize particular antigen (may bind to different epitopes)
mAbs –> from cloned B cells, so only one specific antibody, not produced naturally, can be produced by hybridoma (fusing B cell with cancer cell line)

Question 4

What are 5 different applications for antibodies?

Answer 4

1) ELISA –> Immobilize antigen in well, add antibodies, add antibody that binds to first antibody, 2nd antibody has enzyme that reacts to produce coloured product to indicate amount of antigen present
2) Immunoblotting –> electroblot protein bands from SDS-PAGE onto polymer membrane, then do same as ELISA so that only protein bands with antigen appear
3) Labeling proteins to visualize –> attach flourescent antibodies
4) Immunoprecipitation –> type of affinity chromatography, use ligand to elute bound protein
5) Co-immunoprecipitation –> antibody can pull down entire protein complex, then separate complex by SDS-PAGE or 2D gel electrophoresis (analytical technique)

Question 5

What are 2 types of mass spectroscopy?

Answer 5

mass spec determines precise mass
1) MALDI-TOF
2) ESI
proteins ionized, move through vacuum by electric field according to charge to mass ration (smaller proteins move faster) time taken to reach detector can measure mass