methods for analysing protein protein interactions Flashcards
why is it important to be able to measure protein-protein interactions?
intracellular signalling relies on protein-protein interactions to bring about a response within the cell
which methods can be used to investigate protein-protein interactions?
affinity chromatography
co-immunoprecipitation
what is affinity chromatography used for?
when you want to identify the proteins that interact with protein X
what is an affinity tag?
a small molecule or protein which binds to the ligand with high specificity and affinity
what is sepharose?
a highly crosslinked version of agarose with a reactive group on the surface, used for covalently binding the ligand for the affinity tag
give some examples of affinity tags and name their ligands
glutathione S transferase - ligand is glutathione
histidine - ligand is nickel
how do you identify binding partners when performing affinity chromatography?
if you have the antibodies - can use antibodies to separate, then run on SDS-PAGE and perform western blot
when performing affinity chromatography, how do you know the proteins that are bound have bound via protein X and not GST?
incubate the cell extract with an excess of glutathione-sepharose beads. elute glutathione and analyse equate with SDS-PAGE and a western blot
how can you tell when non-specific binding has occurred in affinity chromatography?
analysis of elute will show a band when cell extract and GST are incubated together
how can you identify proteins from affinity chromatography if you don’t have antibodies for them?
mass spectrometry. analyse peptide fragments according to mass in order to determine sequence
what are the advantages of identifying proteins by mass spectrometry?
- 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
where does trypsin cut?
after arginine or lysine residues except if followed by proline
what are the limitations of mass spectrometry?
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
what is immunoprecipitation?
the use of a single antibody to isolate a protein of interest from a complex mixture of proteins
why is protein A used in immunoprecipitation experiments?
protein A is found in bacterial cell walls and has high affinity for the Fc region of an antibody
what is pre-clearing the lysate?
where a cell lysate is pre-incubated with protein-A sepharose beads
what is the importance of pre-clearing the lysate?
important control to ensure that interactions are between protein X and binding proteins and not bound non-specifically
aside from pre-clearing the lysate, what is an important control for immunoprecipitation reactions?
running a parallel reaction wit an antibody which does not recognise the antigen being studied
what is different about Co-IP?
the antibody used for immunoprecipitating protein X will also pull down associated proteins e.g. protein Y
what is an epitope tag?
short sequences of amino acids which bind to the N or C terminus of the target protein
when is an epitope tag useful?
if an antibody for a target protein is not available
why would you heat immunoprecipitates in a sample buffer prior to performing a western blot?
to separate complexes, break disulphide bonds and coat the proteins with a negative charge
what controls can you use to show that interacting proteins interact with each other specifically?
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
