Exam 5: Lecture 3

Question 1

Yeast 2-Hybrid Assay

Answer 1

• detects protein-protein interactions
• two proteins tested for ability to interact with each other
• one protein fused to GAL4 binding domain and other to activation domain
• plasmids containing DNA sequences for these two chimeric proteins along with plasmid with UAS-lacZ reporter construct are simultaneously transformed into yeast cells
• expression of the fusion proteins induced and yeast cells screened for presence of beta-galactosidase enzyme

Question 2

Interactions in Yeast 2-Hybrid Assay

Answer 2

• if two proteins (X and Y) interact GAL4 activation domain is brought into contact with RNA Pol II which transcribes lacZ gene
• if that happens, yeast cells turn blue
• if two proteins don’t interact then GAL4 activation domain will not be in correct position to stimulate RNA Pol II and remain white

Question 3

Why Yeast 2-Hybrid Assay

Answer 3

• can test ability of just two proteins to interact
• can screen all proteins encoded by the genome to interact with one particular protein of interest
• in later scenario, library of cDNAs is cloned into plasmid containing GAL4 AD
• library transformed into populations of yeast cells
• cells then co-transformed with plasmid containing the gene of interest fused to GAL4 BD
• yeast colonies are screened for putative protein-protein interactions

Question 4

Co-Immunoprecipitations of Proteins

Answer 4

• detects protein-protein interactions
• cells transfected with plasmids that encode two proteins being tested
• proteins extracted from cells and antibody recognizing one of the two proteins (red) is added to lysate
• antibody-protein complex is purified
• antibody-protein lysate run onto a gel and proteins transferred to filter paper
• antibodies against both proteins are added to filter paper (immunoblot step)
• can then determine if antibodies can detect either of the two proteins

Question 5

Results of Co-Immunoprecipitations Assay

Answer 5

• if two proteins (red and green) form complex then two bands seen on filter paper
• red protein is isolated since it came through IP step
• green protein detected in IB step because it’s part of the complex
• if two proteins don’t interact then only one band shows up (red protein isolated in first step)
• since green protein did not interact with red it’s not loaded onto gel

Question 6

Split YFP Assay

Answer 6

• in vivo assay that can be used to determine if two proteins physically interact in cell
• YFP is variant of GFP that emits light in different wavelength in GFP
• can be split into two halves-neither half will glow yellow
• if both halves are allowed to interact then YFP is reconstructed and it will glow yellow
• attach two proteins to each half of YFP if they interact the halves come together and glow, if they don’t interact it won’t glow.

Question 7

Western Blot

Answer 7

• determines if particular protein is present within a cell population, a tissue, or organ
• can be used to compare wild type and mutant samples from different developmental time points

Question 8

Lysate (Western Blot)

Answer 8

• methods exist for isolating proteins from cells and tissue (protein slurry=lysate)
• entire protein lysate loaded onto polyacrylamide gel and separated by size
• protein lysate transferred to nitrocellulose membrane to which they are affixed using cross-linking reagent
• chemiluminescent labeled antibodies are incubated with filter and used to detect protein of interest

Question 9

Protein Detection

Answer 9

• there are chemicals that can detect all proteins without any regard to specificity
• also possible to use labeled antibodies to detect specific proteins
• former case all proteins within a lysate will be visualized
• later case only subset of proteins detected
• starts with primary antibody that recognizes protein of interest
• secondary antibody is labeled with chemiluminescent tag is then used to bind to primary antibody