Question

1. Secondary antibody is dilutes by using
   1. 5 & nonfat dry milk               C. 5 % BSA
   2. 5 % SDS                                D. 5 % PBS

2. For protein detection in the completed Western blot, which instrument is used?
   1. Flow cytometry                     C. Bio imaging system
   2. UV illuminator                       D. Gel documentation system

3. Correct assembly of the immunoblot sandwich from CATHODE to ANODE is

1. Gel             2. Filter paper           3. Sponge     

4. PVDF or Nitrocellulose membrane

A. 2, 3, 4, 1, 3, 4                               C. 3, 2, 1, 4, 2, 3

                        B. 1, 4, 3, 2, 4, 1                               D. 4, 3, 2, 1, 2, 3

4. Western blotting combines several techniques to resolve and detect a specific protein. Below are several steps in the process of running such an experiment, but they are out of order. Put the steps into the correct order.
5. 1. Electrotransfer protein onto a porous membrane
   2. Wash step to remove unbound protein
   3. Incubate with primary antibody specific for antigen or interest
   4. Perform chemiluminescent detection with appropriate reagents
   5. Electrophorese protein sample by SDS gel
   6. Incubate with secondary antibody

1. 5, 1, 3, 2, 6, 2, 4
2. 4, 2, 6, 2, 3, 1, 5
3. 5, 1, 2, 3, 6, 2, 4
4. 3, 2, 6, 2, 5, 1, 4
5. 1, 5, 3, 2, 6, 2, 4
6. None of these are correct

Answer 1

1. D.) 5% PBS. PBS is phosphate buffer saline. It is a isotonic buffer used in biological applications like washing cells, transportation of tissues, dilution. It is used to maintain constant pH of 7.4. The osmoregulatory and ion concentration usually match with human body.

2. D.) Gel Documentation system. The gel Documentation system is used for the detection of protein in the completed western blot. It is based on the principal of flouroscence With flouroscence staining of nucleic acid and proteins used in molecular biology. It is used for imaging or documentation of nucleic acid and proteins suspended with polyacrylamide gel or agarose gel.

3. C.) 3,2,1, 4,2,3.

Correct assembly of immuno blot sandwich from cathode to anode is.

3. Sponge(Cathode -)

2. upper filter paper stack,

1. gel,

4. nitrocellulose membrane/ PVDF,

2. lower filter paper stack,

3. sponge ( Anode+).

4. F.) None of these steps are correct.

The correct steps are:- ( 6,1,2,3,5,4)

6. Electrophoresis protein sample by SDS gel

1. Electrotransfer protein on porous membrane

2. Wash to remove unbound protein

3. Incubate with primary antibody specific for antigen of interest.

5. Incubate with secondary antibody

4. Perform chemiluminescence detection with appropriate reagent.