Question

Please answer 4 post lab questions thank you

Reagents, Supplies and Equipment

1X Blotting buffer pH 8.3 (25 mM Tris, 192 mM Glycine, 20% ethanol)

1X Blocking buffer (5% nonfat dried milk powder in 1X PBS and 0.025% Tween 20)

Wash buffer (1X PBS and 0.025% Tween 20)

Nitrocellulose membrane

Blotting paper

Fiber pads

Western Blotting cassette

Primary antibody – specifically bind only to myosin light chain protein

Secondary antibody

HRP color detection substrate (4-chloro-1-naphthol (4CN))

Mini-PROTEAN Tetra gel electrophoresis system

Microcentrifuge tubes and microcentrifuge tube rack

Pipets and pipet tips

Procedure

Part A – Western Blotting

1. Using a ruler, chop the top off the gel. Locate the lowest band on the protein ladder, chop the portion of the gel below this band
2. Equilibrate the gel in blotting buffer for 15 minutes on a rocking platform.
3. Soak fiber pads thoroughly in blotting buffer.
4. Mark the bottom right corner of the nitrocellulose membrane with a ball point pen
5. Make the blotting sandwich (Figure 1):
   1. Add enough blotting buffer to a container and insert the plastic cassette with red side down
   2. Lay a wet fiber pad on the black side of the cassette
   3. Lay one wet blotting paper and roll out air bubbles with a large pipet tip
   4. Lay wet nitrocellulose membrane on the gel and roll out air bubbles
   5. Lay gel squarely on the nitrocellulose membrane and roll out air bubbles
   6. Lay one wet blotting paper on the gel
   7. Lay a wet fiber pad on top of the blotting paper
   8. Close the cassette and clap together with a white clip

Figure 3 – Schematic of setting up transfer apparatus

6. Set up the Tetra blotting module with the black side of the cassette next to the black side of the Tetra blotting module. Add a frozen blue cooling unit and fill with blotting buffer up to the white clip.
7. Place lid on tank and blot at 100V for 30 minutes or 20V for 2.5 hours.
8. Dismantled the sandwiches and examine the nitrocellulose membrane. The protein standard should be visible on the membrane
9. Put the membrane in in a small tray or container, pour the blotting buffer over the membrane, cover the container and put in the refrigerator.

Part B – Immunodetection

1. Take the tray out of the refrigerator
2. Pour off the blotting buffer and add enough blocking buffer (~10 – 20mL) to cover the membrane
3. Place the tray on a rocker for 30 minutes
4. Pour off blocking solution
5. Add 10 mL anti-myosin primary antibody solution to the tray and place the rocker for 30 minutes at room temperature
6. Pour off the anti-myosin primary antibody solution back into its original container. The antibody can be re-used
7. Add enough wash buffer to cover the membrane. Quickly rinse the membrane and pour off the buffer.
8. Add another set of wash buffer to the membrane and place on rocker for 5 minutes.
9. Pour off the washing buffer
10. Add 10 mL secondary antibody solution to the membrane and place on rocker for 30 minutes.
11. Pour off the secondary antibody solution
12. Rinse the membrane as in step 7.
13. Add another set of wash buffer to the membrane and place on rocker for 5 minutes.
14. Pour off wash buffer
15. Add 10 mL of HRP color detection reagent, place on rocker and allow at least 10 minutes for bands to develop
16. Once the bands have developed, discard the detection reagent and rinse the membrane twice in distilled water and pat it gently between sheets of paper towel
17. Take a photograph of the membrane for your result.

Post-lab Questions

1. What is in the blocking solution?

2. What is Tween-20?

3. What is the enzyme conjugated to the secondary antibody?

4. What is the substrate for the enzyme in question 3?

Answer 1

1) Blocking solution : The blocking solution is made with 5% BSA or nonfat dried milk diluted in TBST (Tris Buffer saline with Tween 20) . The Blocking solution is needed to reduce the background staining by preventing the antibodies to bind nonspecifically.

2) Tween 20 : Tween 20 is a non ionic surfectant that is used as a detergent in many laboratory works. It is stable and relatively non toxic in nature and it is mostly added to buffers o enhance spreadiing of the reagent and decrease background staining.

3) Enzyme conjugated to the secondary antibody is Horse radish peroxidase or HRP

4) The substrate for the enzyme is 4-chloro-1-naphthol (4CN)