SDS PAGE of Fish Muscle
Aim: SDS PAGE separation of proteins using Bio-Rad Comparative Proteomics kit
Materials:
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1.5 ml screw cap tube
1.5 ml microcentrifuge tube
Laemmli Sample Buffer
Samples of different fish muscles (4 fish samples-cod, tilapia, salmon and
catfish)
Mini-protein TGX gel (4-20%)
Actin/Myosin standards (1 vial each, will act as a positive control)
Buffer Dam (if one gel is running)
1X Tris-glycine-SDS gel running Buffer (this comes in a 10X conc)
50 µl of β-mercaptoethanol (BME) is added/950 µl of sample buffer to a final
concentration of 5% (for best results do not store protein samples with BME at 200C
10) Staining trays
11) Water bath at 950C
12) Power supply
13) Rocking platform
14) Gel Doc Go-to analyze stain free gels
Flowchart of SDS PAGE
Procedure:
1) Cut the samples using a scalpel and tweezers into approximately 2 mm cubes
and transfer it into the microcentrifuge tube with 250 µl Laemmli sample buffer
2) Flick or shake the microcentrifuge tube to agitate the fish in the sample buffer
3) Incubate the tube for 5-7 mins at room temperature.
4) Following room temperature incubation, carefully transfer the buffer from the
microcentrifuge tube into the screw cap tube by pouring. Be careful to not
transfer any of the fish muscle tissue.
5) Firmly attach the cap on the tube. Heat the tube for 5 mins at 95 0C on a water
bath.
6) This is a good stop point for the laboratory. You can store the protein extracts
at -200C for up to 6 months.
Electrophoresis of Samples:
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Prepare the mini protein TGX gel by removing it from its package
Pull the tape off the bottom of the gel
Next, remove the comb.
The gel has 2 plates: a short plate on one side and a long plate on the other side
Setting up the vertical electrophoresis unit
1) Remove the electrode assembly from the tank and open
2) Open the green clamps and place a gel into the electrode assembly with the
short plate facing inward
3) Place a second gel on the opposite side of the assembly. If only one gel is run,
use a buffer dam to replace the second gel.
4) Push the gels toward each other making sure they are flush against the notch
green gaskets and that the short plates sit just below the notches of the gasket.
5) Close the green clamps over the gels.
6) Lower the electrode assembly into the tank on the side of the tank with the plastic
tab
7) Make sure to match the red to the red and black to the black
8) Lower the second assembly in the same manner
9) Fill the chamber of the assembly with 1X TGS electrophoresis buffer so the short
plates of the gel are covered.
10) Add buffer to the outer tank up the topmost line.
11) The gels are now ready to load.
12) Heat the fish samples and the actin and myosin standard to 950C for 2-5 mins.
13) Load 5 µl of the protein standard marker in the first well.
14) Load the samples in the remaining lanes.
15) Load the actin and myosin standards
16) Place the lid on the electrophoresis chamber and connect the electrical leads into
the power supply in the proper orientation (red to red and black to black)
17) Turn on the power and set the voltage to 200V and timer to 30 mins.
18) Watch the progression of the blue tracking dye of the Laemmli buffer and the
separation of the standards.
19) When electrophoresis is complete, turn off the power, remove the lid, remove the
electrode assembly, and pour the buffer from the inner chamber back into the
tank.
20) Release the green clamps and remove the gel cassette from the electrode
assembly.
21) Remove the gel from the cassette using the opening key.
22) Carefully remove the gel and transfer it to a staining tray.
Loading samples on the gel (a guide)
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1. protein standard marker -10 µl
2. Actin standard+Laemmli Buffe+1 µl of BME- 5 µl+ 5 µl =11 µl
3. Myosin standard +1 µl of BME - 5 µl+ 5 µl =11 µl
4. cod
5. tilapia
6. salmon
7. catfish
8. cod+BME
9. tilapia+BME
10. salmon+ BME
11. catfish+ BME
Gel Analysis:
The image will be viewed and analyzed on the GelDoc Go machine.
Please consult your instructors for the proper use of the machine to visualize and
analyze your gels.