Microbiology 5: Analysis protein-protein interactions

Question 1

2 methods for studying protein-protein interactions

Answer 1

Affinity chromatography

Co-immunoprecipitation

Question 2

basic Steps of affinity chromatography

Answer 2

• add affinity tag which bind to ligand with high affinity and high specificity
• Matrix with ligand for affinity tag attached binds to protein-affinity tag complex

Question 3

Examples of 2 affinity tags and their ligands

Answer 3

(Tag) Glutathion-S-Transferase
- (Ligand) Glutathione

(Tag) Histidine
- (Ligand) Nickel

Question 4

Describe an experiment using affinity chromatography as a method of purifying proteins

Answer 4

Experiment for ‘protein X’

Recombinant DNA technique is used to fuse the gene for protein X with Glutathione-S-Transferase (GST)

Insertion into cell -> translation of GST-X fusion protein

Protein purified using GLUTATHIONE beads in affinity column matrix

Wash and collect proteins from affinity column

Perform SDS-PAGE followed by western blot analysis
(or MASS Spec)

Question 5

Steps of mass spectrometry

Answer 5

• Protein digested with enzyme e.g. Tripsin
• Protein sample is ionized
• Electrical field accelerates ions
• Lightest ions arrive at detector first
• Laser triggers a clock

Results in peptide mass fingerprint

Question 6

What is immunoprecipitation?

Answer 6

use of a specific antibody to isolate protein of interest from a mixture of proteins e.g. cell extract

Question 7

Basic steps of immunoprecipitation

Answer 7

• Lyse cell to create cell extract
• Add antibody -> antibody binds to target protein with high affinity
• Add protein A-sepherose beads -> these bind to antibody
• newly formed complex sediments at bottom of tube
• wash + add SDS + mercaptoethanol
• can perform western blot/ enzyme assay depending on target protein function

Question 8

advantages of mass spectrometry

Answer 8

Can determine molecular weights of peptide fragments with great accuracy

Highly sensitive technique which requires very small amounts of sample

Question 9

2 proteins, X and Y. What differs with immuno and co-immunoprecipitation methods?

Answer 9

co-immuno remains same if Y is associated unless antibody for X is not available

If antibody is not available, recombinant DNA techniques can be used to fuse an Epitope tag (10-15aa’s long) to end of target protein

Commercial antibodies available to bind to different epitope tags