Test 8

Question 1

What is Western blotting used for?

Answer 1

identification and quantification of protein antigens

Question 2

What are the steps in Western blotting?

Answer 2

1. sample preparation
2. gel electrophoresis
3. transfer
4. blocking
5. detection

Question 3

How are proteins separated according to in Western blot?

Answer 3

molecular weight
* proteins are separated into bands within each lane formed under each well
* compared to a commercially prepared mixture with defined molecular weight electrophoresed in one lane

Question 4

what is the isoelectric point?

Answer 4

The pH at which the molecule is electrically neutral

Question 5

What gels are used in Western blot?

Answer 5

Polyacrylamide and 2D

Question 6

What are proteins transferred onto in Western blot?

Answer 6

nitrocellulose; polyvinylidene difluoride

Question 7

Why must blocking be used in Western blot?

Answer 7

antibodies used for detection of target protein would bind to membrane, leads to clearer results and eliminates false positives

Question 8

What solutions are used for blocking in Western blot?

Answer 8

bovine serum albumin or casein, detergent

Question 9

What happens during blocking in Western blot?

Answer 9

protein in dilute solution attaches to the membrane in all places where target proteins have not attached

Question 10

What secondary immunologically reagents are used in detection phase of Western blot?

Answer 10

antiglobulin or protein A labelled with enzyme

Question 11

What types of substrate can be used in detection phase of Western blot?

Answer 11

Colorimetric, photometric and chemiluminescent