Question

Reagents, Supplies and Equipment

Laemmli sample buffer containing Dithiothreitol (DTT) at 10 mg/mL

10X tris-glycine-SDS (TGS) electrophoresis buffer

Bio-Safe Coomassie stain for proteins

Actin and myosin standard

Precision Plus Protein Kaleidoscope pre-stained standard

Mini-PROTEAN pre-cast polyacrylamide gels

Mini-PROTEAN Tetra gel electrophoresis system

Microcentrifuge tubes and microcentrifuge tube rack

Heat block at 95^oC

Pipets and pipet tips

Procedure

Part A – Protein Extraction

1. Using a spatula, put a small amount of fish or meat into a microtube. Weight the tube to make sure the mass is similar (~50 - 100 mg).
2. Add 250 µl of Bio-Rad Laemmli sample buffer to each microtube.
3. Flick the microtubes 15 times to agitate the tissue in the sample buffer.
4. Incubate for 5 minutes at room temperature.
5. Carefully transfer the buffer by pouring from each microtube into a labeled screwcap microtube. Do not transfer the fish!
6. Proceed to part B. Once the gel apparatus has been set up, proceed to step 7 below.
7. Mix 20 µL of your purified GFP with 20 µL Laemmli sample buffer in a screwcap microtube.
8. Heat the meat/fish and the GFP samples in microtubes for 5 minutes at 95°C.
9. Carefully take out the screwcap tube and prepare for SDS-PAGE electrophoresis

Part B – SDS-PAGE Electrophoresis

Two gels will be run for each of your samples. One gel will be stained with Coomassie blue stain and the other will be used for Western Blotting.

Set up Mini-PRPTEAN Tetra gel apparatus

1. Take out two pre-cast acrylamide gels from the packages.
2. Remove the comb from the pre-cast gel.
3. Place gel cassettes into the electrode assembly with the short plate facing inward.
4. Push both gels toward each other, making sure they are against the green gaskets that are against the green gaskets that are built into the clamping frame; make certain that the short plates sit just below the notch at the top of the green gasket. Slide the green arms of the clamping frame over the gels, locking them into place.
5. Lower the electrode assembly with the gels in it into the mini tank on the side of the tank with the plastic tabs. Make sure that the red banana plug goes on the side of the tank with the red oval.
6. Completely fill the inner chamber with 1X TGS electrophoresis buffer, making sure the buffer covers the short plate (~150 mL).
7. Fill mini tank with approximately 700 mL of 1X TGS electrophoresis buffer until the buffer reaches the 2 gels line on the tank.

Electrophoresis

8. Once the fish samples are finished heating at 95^oC for 5 minutes (step 7, part A), load the sample into the gels as follow:

 

Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6	Lane 7	Lane 8	Lane 9	Lane 10
Protein Standard	Student A Sample 1	Student A Sample GFP	Student B Sample 1	Student B Sample GFP	Student C Sample 1	Student C Sample GFP	Student D Sample 1	Student D Sample GFP	Actin and myosin standard

 

9. Electrophorese for 30 minutes at 200V in 1X TGS electrophoresis buffer or until blue dyes at the bottom of the gel.
10. After electrophoresis, remove gel from cassette and wrap with paper tower and put into zip bag.
11. Pour enough 1X TGS electrophoresis buffer into the zip bag to keep gel from dying up.
12. Store gel at 4^oC.

Part C – Staining and De-staining

1. Remove gel from cassette and place gel in a staining tray.
2. Rinse gels in tap water 3 times for 5 minutes each. Rock gently by hand.
3. After the last rinse, pour off water and add 50 mL of Bio-Safe Coomassie blue stain. Stain gels for at least 1 hour with gently shaking or rock gently by hand.
4. Discard stain and de-stain gel in a large volume of water overnight. Cover the container with plastic wrap and leave at room temperature to de-stain.
5. The instructor will have the water removed once and the de-stained gel will be kept in water at 4^oC until the next class.
6. Remove the de-stained gel from the water and put it on a white drying paper.
7. Photograph the gel for analysis.
8. Using the protein standard in lane 1, and actin/myosin standard in lane 10, identify actin and myosin in your samples.

 

Post-lab Question

1. How do we visualize the protein profile of the sample after SDS-PAGE?

 

 

2. How do you tell if your purified GFP is pure after staining the SDS-PAGE?

 

 

3. Why do we use nitrocellulose membrane for the transfer of protein from SDS-PAGE gel to blot?

 

 

4. Which protein will the primary antibody used in this lab bind to?

Answer 1

a) Visualization of protein profile after SDS PAGE gel is achieved by coomasie blue staininteractioning and Destaining. The Coomassie dyes (R-250 and G-250) bind to proteins on the SDS PAGE gel through ionic interactions between dye sulfonic acid groups and positive protein amine groups and also via van der waals interaction. Upon Destaining the gel, the blue colour gets off from the parts where there is no protein, leaving only the protein bands to be stained.

b) After staining and destaining the SDS PAGE gel, we can tell whether the GFP sample purified is pure Or not. GFP sample will have a specific molecular weight and the protein will appear on the gel exactly corresponding to the same molecular weight as the ladder. If there are other bands along with the GFP band, then the protein is impure. And if there is single band of GFP, then the protein is pure.

c) After gel electrophoresis, the gel is transferred to a membrane using electric field perpendicular to the surface of the gel. Nitrocellulose membrane is used for this purpose of transferring because of it's high affinity for protein and its retention abilities.

d) The primary antibodies are the ones which bind to the protein of interest on the membrane.

Anti-myosin antibody will bind to the myosin band.

Anti-actin antibody will bind to the actin band

Anti-GFP antibody will bind to the GFP band.