Polvi lec #6

Question 1

What does in vivo mean?

Answer 1

It means studying proteins within their biological setting

Question 2

What does in vitro mean?

Answer 2

It means studying proteins isolated from their biological setting

Question 3

What is FRAP?

Answer 3

is fluorescence recovery after photobleaching and is a invivo technique to study the movement of membrane components

Question 4

How does FRAP work?

Answer 4

You label the membrane component with a fluorescent dye by putting a fluorescent tag on an antibody that recognizes the protein, you then photobleach a portion of the protein and then monitor the reappearance of the flourescence

Question 5

What does the rate of recovery of fluorescence tell you about a protein?

Answer 5

It measures the rate of diffusion of the fluorescently labelled protein

Question 6

How do you isolate membrane proteins in vitro?

Answer 6

you can lyse the cells, isolate for certain proteins and then visual through gel electrophoresis

Question 7

Explain the procedure of isolating peripheral, transmembrane, and lipid anchored proteins?

Answer 7

lyse the cells and centrifuge the sample to get supernatant and pellet (which has membrane material)
Isolate peripheral membrane proteins: use high salt to disrupt the noncovalent interactions the proteins amino acids have with the membrane, centerfuge and in pellet 2 you have insoluble material and supernatant 2 you’ll have peripheral membrane proteins
Isolate transmembrane proteins: Use a detergent as they are amphipathic and will interact with nonpolar regions of protein and have polar heads that will keep it soluble in aq solution, centerfuge and you’ll get transmembrane proteins in supernatant and the remaining membrane material in the pellet.
Isolating GPI anchored proteins: Use PI-PLC to cleave PI bond between leaflet and oligosaccharide chain connected to protein, centerfuge and GPI anchore dproteins will be in supernatant

Question 8

Why can’t you use detergent to isolate GPI anchored proteins?

Answer 8

Because they are found in detergent resistant portions of the membrane

Question 9

What is polyacrylamide gel electrophoresis?

Answer 9

Proteins migrate through a gel matrix made of polyacrylamide (cross linked acylamdie polymers)

Question 10

How does PAGE work?

Answer 10

Denature proteins thorugh SDS, this unfold them and breaks bonds making it neg throughout
mix proteins with tracking dye and load onto polyacrylamide gel
stain proteins with coomassie blue dye
load into gel with ladder and then apply electric current

Question 11

What does PAGE tell us about proteins?

Answer 11

their size and their expression, if it doesn’t travel far it’s big and if it’s high in expression it’s a thicker band