Practical 2 - SDS-PAGE and Western Blotting

Question 1

insulin is the stimulus for trafficking to the plasma membrane. If disruption via CRISPR/Cas9 has been successful will there be trafficking of the protein to the plasma membrane?

Answer 1

no

Question 2

what is the function of SDS in this practical?

Answer 2

to denature and add a negative charge evenly over the proteins

Question 3

what is the blocking agent normally made of in this experiment?

what is its function?

Answer 3

made of milk powder, casein or BSA

prevents non-specific binding of antibody to membrane

Question 4

if the adipocyte has been given insulin, where would you expect to find the GLUT4 protein within the cell?

why?

Answer 4

on the cell membrane, so that glucose can be transported out of the blood stream and into cells to reduce blood sugar levels back to normal

Question 5

name a viral disease in which western blotting can be used to confirm the presence of the disease

Answer 5

HIV or BSE

Question 6

using a whiteboard, you have 50ml of solution. it needs diluting 1 in 1000. How many microliters of solution will be used?

Answer 6

if 1ml in 1000ml
then 0.1ml in 100ml
then 0.05ml in 50ml

so therefore need 0.05ml or 50 microliters of solution

Question 7

using a whiteboard, if you have 50ml of solution and it needs diluting 1 in 5000, how many microliters will you need?

Answer 7

1ml in 5000ml
=0.2 in 10000ml
=0.02 in 100ml
=0.01 in 50ml

so 0.01ml or 10 MICROLITERS

Question 8

what is the function of the Tween 20 used in the blotting solutions?

Answer 8

to act as a solubilizing agent and as a blocking agent to improve sensitivity of antibody detection

Question 9

what is the consequence of using too much Tween20 in the blotting solutions?

Answer 9

it could wash away the bound antibody or antigen from the blot

Question 10

what does the primary antibody bind specifically to?

Answer 10

the protein of interest only

Question 11

what does the secondary antibody bind to?

why is it used?

Answer 11

the primary antibody

used to amplify signal and increase sensitivity

Question 12

why is the secondary antibody coupled to an enzyme?

Answer 12

to catalyse a reaction which results in a coloured/chemiluminescent band = shows location of protein

Question 13

using a whiteboard, if you had a 10x stock of transfer buffer how would you calculate the amount of water required to make up a 1x working solution?

Answer 13

you would dilute the stock solution by adding 9 parts of water for every 1 part of the stock solution

Question 14

using a whiteboard, work out how you would make up 25ml of 1 in 1000 primary antibody?

Answer 14

divide 25 by 1000 to get the volume of antibody:
= 0.025ml

minus 0.025 from 25 to give you how much water you would need:
= 24.975ml

this gives you a 1mg/ml primary antibody solution.

Question 15

using a whiteboard, work out how you would make up 40ml of 1 in 5000 secondary antibody?

Answer 15

divide 40 by 5000 to give the ml of antibody needed:

= 0.008ml

minus 0.008 from 40 to give ml of water needed for dilution:

= 39.992 ml

Question 16

what is the function of mercaptoethanol/DTT?

Answer 16

to reduce disulfide bridges between cysteine residues

Question 17

Western blotting is a powerful and useful technique. First proteins are separated by 1.____-PAGE by molecular weight. The proteins are denatured before running on the gel by heating them in Sample Buffer. The Sample Buffer contains 2.____, which acts as a buffer to maintain pH, 3.____ to denature and unfold the protein, and 4.____ which breaks disulphide bonds and therefore aids denaturation. Once the gel is run the proteins are transferred to a nitrocellulose membrane and probed with antibodies. One critical step in this process is the blocking of extra protein binding sites on the nitrocellulose sheet. These extra protein binding sites can be blocked with 5.____ or milk in the buffer.

Answer 17

1 - SDS
2 - Tris
3 - SDS
4 - DTT
5 - BSA/milk powder/ casein