LECTURE 6 (Immunoprecipitation)

Question 1

What is Immunoprecipitation?

Answer 1

A technique in which an antibody specific for one protein antigen in a mixture of proteins is used to identify a specific antigen

Question 2

How does Immunoprecipitation work?

Answer 2

1) Antibody is added to a protein mixture and staphylococcal protein A (or protein G) covalently attached to agarose beads is added to the mixture
2) Fab portions of the antibody bind to the target protein and the Fc portion of the antibody is captured by the protein A/protein G on the beads
3) Unwanted proteins that do not bind to the antibody are removed by washing the beads (by repeated detergent addition + centrifugation)

Question 3

How can specific proteins bound to antibodies be eluted from the beads and dissociated from the antibody?

Answer 3

Use of a harsh denaturant + proteins are separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)

Question 4

How can proteins be detected after electrophoresis?

Answer 4

• Staining the polyacrylamide gel with a protein stain
• Western blot analysis

Question 5

What can happen if the original mixture contained radioactively labeled proteins?

Answer 5

Specific proteins immunoprecipitated by the antibody may be revealed by AUTOFLUOROGRAPHY/AUTORADIOGRAPHY with protein bands being captured on x-ray film placed on dried SDS-POLYACRYLAMIDE GEL containing separated proteins

Question 6

What is Immuno-affinity chromatography?

Answer 6

A purification method that relies on antibodies attached to an insoluble support to purify antigens from a solution

Question 7

What happens to unbound molecules in immuno-affinity chromotography?

Answer 7

• Unbound molecules are washed away
• Bound antigen is elute by changing the pH or by exposure to high salt or other chaotropic conditions that break antigen-antibody interactions