methods for analysing protein protein interactions

Question 1

why is it important to be able to measure protein-protein interactions?

Answer 1

intracellular signalling relies on protein-protein interactions to bring about a response within the cell

Question 2

which methods can be used to investigate protein-protein interactions?

Answer 2

affinity chromatography

co-immunoprecipitation

Question 3

what is affinity chromatography used for?

Answer 3

when you want to identify the proteins that interact with protein X

Question 4

what is an affinity tag?

Answer 4

a small molecule or protein which binds to the ligand with high specificity and affinity

Question 5

what is sepharose?

Answer 5

a highly crosslinked version of agarose with a reactive group on the surface, used for covalently binding the ligand for the affinity tag

Question 6

give some examples of affinity tags and name their ligands

Answer 6

glutathione S transferase - ligand is glutathione

histidine - ligand is nickel

Question 7

how do you identify binding partners when performing affinity chromatography?

Answer 7

if you have the antibodies - can use antibodies to separate, then run on SDS-PAGE and perform western blot

Question 8

when performing affinity chromatography, how do you know the proteins that are bound have bound via protein X and not GST?

Answer 8

incubate the cell extract with an excess of glutathione-sepharose beads. elute glutathione and analyse equate with SDS-PAGE and a western blot

Question 9

how can you tell when non-specific binding has occurred in affinity chromatography?

Answer 9

analysis of elute will show a band when cell extract and GST are incubated together

Question 10

how can you identify proteins from affinity chromatography if you don’t have antibodies for them?

Answer 10

mass spectrometry. analyse peptide fragments according to mass in order to determine sequence

Question 11

what are the advantages of identifying proteins by mass spectrometry?

Answer 11

• can determine molecular weights of peptide fragments with great accuracy (0.1-0.01%)
• highly sensitive technique - does not require large amounts of a sample

Question 12

where does trypsin cut?

Answer 12

after arginine or lysine residues except if followed by proline

Question 13

what are the limitations of mass spectrometry?

Answer 13

only identifies peptides which are approximately 8-30 amino acids long. peptides can be too small or too large to be detected. mass spec will never give 100% sequence coverage

Question 14

what is immunoprecipitation?

Answer 14

the use of a single antibody to isolate a protein of interest from a complex mixture of proteins

Question 15

why is protein A used in immunoprecipitation experiments?

Answer 15

protein A is found in bacterial cell walls and has high affinity for the Fc region of an antibody

Question 16

what is pre-clearing the lysate?

Answer 16

where a cell lysate is pre-incubated with protein-A sepharose beads

Question 17

what is the importance of pre-clearing the lysate?

Answer 17

important control to ensure that interactions are between protein X and binding proteins and not bound non-specifically

Question 18

aside from pre-clearing the lysate, what is an important control for immunoprecipitation reactions?

Answer 18

running a parallel reaction wit an antibody which does not recognise the antigen being studied

Question 19

what is different about Co-IP?

Answer 19

the antibody used for immunoprecipitating protein X will also pull down associated proteins e.g. protein Y

Question 20

what is an epitope tag?

Answer 20

short sequences of amino acids which bind to the N or C terminus of the target protein

Question 21

when is an epitope tag useful?

Answer 21

if an antibody for a target protein is not available

Question 22

why would you heat immunoprecipitates in a sample buffer prior to performing a western blot?

Answer 22

to separate complexes, break disulphide bonds and coat the proteins with a negative charge

Question 23

what controls can you use to show that interacting proteins interact with each other specifically?

Answer 23

carry out IP with non-specific antibody - this will not pull down protein of interest and there will not be a band for it on SDS-PAGE