Western Blot

Question 1

What does western blot do

Answer 1

Indicates steady state levels of a specific protein at a given time

Question 2

Step 1

Answer 2

Treat cells as desired

Question 3

If you see an increase in a specific protein, what could be the reason?

Answer 3

• Cell is making more protein (increase in translation)
• Cells are making the same amount of protein but proteins stays around longer
• Cant be more than one protein in the same place because this is specific

Question 4

Step 2

Answer 4

-Extract protein from cells and quantitate

Question 5

Step 3

Answer 5

Add loading buffer of SDS, glycerol, bromophenol blue and beta-mercaptoethanol

Question 6

Step 4

Answer 6

Boil samples

Question 7

Step 5

Answer 7

Run samples on SDS-Page gel

Question 8

Step 6

Answer 8

Transfer proteins to a nitrocellulose membrane

Question 9

Step 7

Answer 9

-Block membrane with BSA or milk

Question 10

Why is milk used to block membrane?

Answer 10

Because Ab are very sticky. The milk binds to membrane where theres no proteins so the Ab doesnt randomly bind to membrane

Question 11

Step 8

Answer 11

Add primary Ab to protein of interest and wash

Question 12

Step 9

Answer 12

Add secondary Ab (to primary Ab) which is conjugated to HRP and wash

Question 13

Step 10

Answer 13

Add chemiluminescent reagent that interact with HRP, giving off light

Question 14

Step 11

Answer 14

Expose to film

Question 15

Loading control

Answer 15

• Strip membrane (remove Ab)
• Select an Ab to a protein that does not change in levels under any conditions (ERK-1 and ERK-2)
• Hybridize to membrane as done previously
• Expose to film
• Denistometry

Question 16

Advantages

Answer 16

• Quick and relatively easy

- Allows the detection of the steady state level of a specific protein

Question 17

Disadvantages

Answer 17

• Only looks at steady state levels of protein

- Ab can be expensive and/or not available