Secretion and Insulin Techniques

Question 1

What is 35 S methionine labelling used for?

Answer 1

Label newly-synthesized proteins
Analysis by different methods e.g., IP followed by PAGE-transfer

Question 2

What are the steps to using digitonin to permeabilise cell membranes?

Answer 2

Aspirate spent medium
Treat cells with trypsin
Harvest cells
Treat cells with digitonin

(Add proteinase K)

Question 3

What is cell lysate PAGE Western blot analysis?

Answer 3

Analysis of proteins from cells
Resolve by molecular weight (SDS-PAGE - nitrocellulose membrane)
Immuno-detection

(sodium dodecyl sulfate–polyacrylamide gel electrophoresis)

Question 4

What are the steps to do PAGE Western blot analysis?

Answer 4

Aspirate spent media
Scrape cells off and lyse
Load proteins onto gel
Resolve proteins
Transfer proteins to nitrocellulose membrane
Incubate with primary antibodies
Incubate with secondary antibodies

Question 5

What are primary and secondary antibodies?

Answer 5

Primary antibodies specifically recognised protein of interest
Secondary antibody recognises primary antibody, usually conjugated with some kind of enzyme that is able to catalyse a substrate for chemilumescence that is detectable by a machine

Question 6

What is immuno-precipitation?

Answer 6

Pulling-down proteins of interest (e.g., because the concentration of the protein is low)
Followed by analysis using PAGE-Western blotting

Question 7

What are the steps to do an immuno-precipitation?

Answer 7

Aspirate spent medium
Collect cells
Lyse cells
Add antibodies conjugated to sepharose beads (to sink to the bottom)
Aspirate supernatant
PAGE followed by Western blot analysis