Module 2 - Proteins - Antibody Assay Techniques

Question 1

Purpose of Western Blotting

Answer 1

detect protein presence

Question 2

Purpose of ELISA

Answer 2

detect protein concentration in a solution

Question 3

Purpose of IHC

Answer 3

detect protein presence and spatial info

Question 4

What method is most accurate for getting the concentration of proteins?

Answer 4

ELISA

Question 5

What method is most accurate for getting spatial info?

Answer 5

IHC

Question 6

Is western blotting quantitative or qualitative

Answer 6

semi of both

Question 7

Is ELISA quantitative or qulitative

Answer 7

quantitative

Question 8

Is IHC quantitative or qualitative

Answer 8

qualitative

Question 9

Which is cheaper? ELISA or Western Blotting?

Answer 9

ELISA

Question 10

Which is easier? ELISA or Western Blotting?

Answer 10

western blotting

Question 11

Which method can detect isoforms

Answer 11

western blotting

Question 12

What are the steps in Western Blotting?

Answer 12

1) Transfer proteins from SDS page gel to nitrocellulose membrane
2) Press gel tightly and send an electrical field (move as proteins are still - charged)
3) Incubate proteins w/ primary antibody
4) Incubate proteins w/ labelled secondary antibody
5) Put membrane in an imaging machine -> scans lasers the colour of the fluorescent molecules

Question 13

What are the steps in ELISA sandwich?

Answer 13

1) Add sample into a 96-well plate
2) Proteins bind to plastic via a non-covalent interaction: plastic has side-chain groups which react with side-chain groups of amino acids of protein sample
3) We coat the antibody directed at our protein of interest (capture antibody)
4) We add the sample with protein of interest - only our antigen of interest will bind to the antibody
5) Wash away the sample so proteins are not bound
6) Add a detection antibody w/ a label

Question 14

How do you amplify Sandwich ELISA?

Answer 14

Add streptavidin which is conjugated to HRP. This will bind to the biotin molecules of the detection antibody (many HRP connected to one detection antibody)

Question 15

ELISA has __ specificity

Answer 15

High (2 antibodies connected to antigen)

Question 16

Why is streptavidin HRP used in ELISA but not IHC?

Answer 16

Not used in IHC because in the tissue used there are lots of other proteins (e.g. membrane, DNA, interstitial tissue) and biotin can lead to a false reaction

Safe in ELISA because there is only one antigen of interest

Question 17

What is the principle of IHC?

Answer 17

we have a protein of interest -> a primary antibody binds to it -> a secondary antibody with a label (HRP or ALEXA) binds to the primary antibody

Question 18

Steps in IHC

Answer 18

1) Make thin sections of 10-30 micrometers of brain

2) Fixation: chemical binding of proteins to tissue is fixed (protein changed a little so antibody can recognize it)

3) Put it on glass -> put this in a solution of primary antibodies which react w/ protein of interest

4) Take it off and put it in a buffer to rinse away proteins that are not bound

5) Amplification: put in a second solution w/ labeled secondary antibody which bind to primary antibody - many secondary antibodies bind to one primary antibody