Exam 5: Lecture 3 Flashcards Preview

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Flashcards in Exam 5: Lecture 3 Deck (9):
1

Yeast 2-Hybrid Assay

-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

2

Interactions in Yeast 2-Hybrid Assay

-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

3

Why Yeast 2-Hybrid Assay

-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

4

Co-Immunoprecipitations of Proteins

-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

5

Results of Co-Immunoprecipitations Assay

-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

6

Split YFP Assay

-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.

7

Western Blot

-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

8

Lysate (Western Blot)

-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

9

Protein Detection

-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