Using one gene to find another

Question 1

What are 4 methods to identify other genes related to our process of interest?

Answer 1

Yeast two hybrid assay, phage display, protein assays, Co-IP

Question 2

How does a yeast two hybrid assay work?

Answer 2

Dependent on an endogenous yeast transcription factor, Gal4p. The technique involves two plasmids, each with one half of Gal4p attached to a protein of interest and a potential interacting partner. If the two proteins interact, the two halves of Gal4p get brought together and activates transcription of a reporter gene

Question 3

What are the 2 plasmids used in a Y2H assay? What is on each one?

Answer 3

Bait: one half of Gal4p fused in frame to your protein of interest, one selectable marker (leu-)
Prey: other half of Gal4p fused in frame to a protein from a library, a different selectable marker (trp-)

Question 4

What is the Bait strain that the bait plasmid is transformed into?

Answer 4

Deletion in the endogenous Gal4 gene, leucine and tryptophan auxotroph, Gal1::LacZ. Plated on complete media -leu

Question 5

What is the prey strain that the prey plasmid was transformed into?

Answer 5

Deletion in the endogenous Gal4 gene, leucine and tryptophan auxotroph, Gal1::LacZ. Plated on complete media -trp

Question 6

What do you do with the bait and prey transformants?

Answer 6

Mate them and plate them on complete media, then replica plate on complete media -leu, -trp, + X-gal

Question 7

How do we tell if the protein of interest and a protein in the library of the prey strain are interacting?

Answer 7

Proteins that interact will bring the two halves of Gal4p together and activate transcription of LacZ, and will be blue. Colonies with no protein interactions are white

Question 8

What are the advantages of Y2H?

Answer 8

Relies on selection, easy to scale up, scores direct interactions, yeast grow really fast, standardized methods for using proteins from any organism we want, not technically challenging, already have the interacting gene cloned

Question 9

What are the disadvantages of Y2H?

Answer 9

Interactions may not be physiologically representative, only binary interactions are seen, might miss proteins that needs post-translational modifications, need a good prey library, need to have YFG already cloned, doesn’t tell us about mutant phenotypes or the overall process

Question 10

How do you identify binding partners through Co-IP?

Answer 10

1. Create a cell lysate and add an antibody attached to a bead against YFP
2. Centrifuge to bring all the antibodies down
3. Take the pellet, elute the proteins off the antibody and run them on an SDS PAGE gel
4. Cut the bands out of the gel and use mass spec to identify them

Question 11

What are the advantages of Co-IP?

Answer 11

Most physiologically relevant, most detailed info on stoichiometry/strength of the interactions, can get multimeric complexes, ensures direct interactions

Question 12

What are the disadvantages of Co-IP?

Answer 12

Technically challenging, requires the most prior knowledge and reagents, no standardized protocols and need to adapt for each organism, doesn’t tell mutant phenotypes or about the overall process

Question 13

How does a protein array work?

Answer 13

Same way as a microarray. Has proteins immobilized on a chip and we add whatever we want to test for interactions to it and get different signals depending on if things bind or not

Question 14

What are the two types of protein arrays?

Answer 14

Analytical: for determining the presence or absence of something
Functional: for characterizing interactions

Question 15

Can protein arrays only detect protein-protein interactions?

Answer 15

No, we can add other stuff to the chip to look for protein interactions with different types of molecules like lipids, DNA, small molecules, etc

Question 16

How do we do a protein array?

Answer 16

1. Get a chip that has every protein from an organism cross linked to the chip
2. Flood the chip with a solution of POI
3. Add a labelled antibody for POI and look for signals

Question 17

Do we have to use antibodies to detect POI on a protein array?

Answer 17

No, we can do other things like adding a biotin tag to POI then probing with streptavidin, or other things like primary then secondary antibody

Question 18

What are the advantages of a protein array?

Answer 18

High throughout and high sensitivity

Question 19

What are the disadvantages of a protein array?

Answer 19

Expensive, not under physiological conditions, only binary interactions, no info on mutant phenotypes or overall process

Question 20

What is phage display?

Answer 20

Generating recombinant phages that will display a fusion protein of one of their coat proteins and your POI

Question 21

How do we do phage display?

Answer 21

Create a cDNA library and put them in a phage vector fused in frame with a phage coat protein. Transform the phage vector into E. coli, then assemble the recombinant phages from that. Then add the phages expressing the recombinant coat proteins to a well coated in POI. Any phages displaying a binding partner will stick to the well, and the others get washed off. Then we can elute the bound phage off POI and sequence it

Question 22

What are the advantages of phage display?

Answer 22

Can manipulate binding conditions, can easily screen large numbers of phages, can easily screen for the impacts of a selected mutations (through site-directed mutagenesis)

Question 23

What are the disadvantages of phage display?

Answer 23

Can be time consuming, no complexes, can miss proteins that need post-translational modifications, folding can be disrupted on the surface of a phage, no info on mutant phenotypes or overall process