Lecture 7- How are proteins studied inside cells?

Question 1

Immunofluorescence

Answer 1

antibody attached to a fluorescent dye

Question 2

Antibody (IgG)

Answer 2

have affinity for the target

hard to make

Question 3

How do you make an antibody?

Answer 3

1. Take a protein
2. Inject it into an animal (ie., rabbit)
3. Wait for the rabbit’s immune system to generate antibodies against it
4. Purify all the antibodies from the rabbit
5. Isolate those that only stick to your favorite proteins

Question 4

Direct immunofluorescence

Answer 4

antibody attached to a fluorescent dye

Nomenclature: “ source anti target antibody”

Question 5

Indirect immunofluorescence

Answer 5

Primary antibody- binds to target
Secondary antibody- binds to primary antibody and the fluorescence is attached to it.

Downside: several steps
Benefits: making the target much more fluorescent

Question 6

Fluorescent protein that is made from jellyfish

Answer 6

GFP

Question 7

Fusion proteins must be tested to ensure they go to their proper cellular location

True or false

Answer 7

True

Question 8

Tagged fusion proteins

Answer 8

Use HA-tag
use 9 a.a sequence

Question 9

3 Methods to eliminate specific proteins inside cells

Answer 9

1. DNA mutation- cells with a non-functioning gene can’t make YFPs
2. Morpholinos- synthetic mRNA-like molecules that bind to target mRNAs and block ribosomes
3. Antibody depletion- depleting the functional proteins in our cell by coding all the proteins with antibodies