Protein Half-Life

Question 1

Western Half Life function

Answer 1

-Determines the half life of a specific protein under specific conditions

Question 2

Step 1

Answer 2

Treat cells as desired

Question 3

What happens right after treatment?

Answer 3

After treatment, the cells will make proteins, some at varying levels

Question 4

Step 2

Answer 4

-After treatment, add puromycin or cyclohexamide to stop any additional translation

Question 5

Step 3

Answer 5

Extract total protein from cells at various times

Question 6

Step 4

Answer 6

Protein concentrations

Question 7

Step 5

Answer 7

Add loading buffer of

• SDS and beta-ME
• Glycerol
• Bromophenol blue

Question 8

Step 6

Answer 8

Boil samples

Question 9

Step 7

Answer 9

Run samples on an SDS Page gel

Question 10

Step 8

Answer 10

Transfer to a nitrocellulose membrane

Question 11

Step 9

Answer 11

-Block membrane with BSA or milk

Question 12

Step 10

Answer 12

-Add primary Ab to protein of interest and wash

Question 13

Step 11

Answer 13

-Add secondary Ab which is conjugated to HRP and wash

Question 14

Step 12

Answer 14

-Add chemiluminescent reagent that interacts with HRP, giving off light

Question 15

Step 13

Answer 15

-Expose membrane to film so we can now see the protein levels

Question 16

Step 14

Answer 16

-Densitometry which gives arbitrary values

Question 17

Is there a loading control?

Answer 17

No, must repeat the experiment

Question 18

Advantages

Answer 18

-Can measure protein half life

Question 19

Disadvantages

Answer 19

• Need more dishes

- More time consuming