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Lab Investigation: Protein Fingerprinting

BIG IDEA: Every cell in an organism has the recipe for every single protein in that organism's body, but what proteins does each cell actually make? During this lab investigation, you will isolate and then separate out the proteins from different bovine tissues (skeletal muscle, heart muscle, liver, etc.) using protein gel electrophoresis. This will create a 'protein fingerprint' of different types of cells from a cow so you can examine whether they make the same or different proteins.

Materials/Equipment Needed

For the class:
• hot water bath (70°C)
• hot water bath or heat block (95°C)
• 2 thermometers
• microwave
• 3% agarose in Tris-Glycine buffer
• Tris-Glycine-SDS buffer
• Coomassie blue stain
• Destain
• Plastic wrap
• 3-4 different bovine tissues

For each student group:
• horizontal gel electrophoresis apparatus, electrodes, power supply
• micropipet with 4 micropipet tips
• 3-4 microfuge tubes with 500 µl sample buffer
• 3-4 empty microfuge tubes
• staining tray
• paper towel or kleenex

Pouring an agarose gel:

1. Get your electrophoresis apparatus and make sure there are black stoppers at both ends of the gel
tray.

2. Make sure one comb is in place at the negative electrode (black end of the gel).

3. Pour melted agarose into the gel space until the gel fills the shallow tray NOT THE WHOLE BOX.
Let the agarose harden, which should take about 10 minutes. Don’t touch/move your gel until it’s hard.
In the meantime, prepare your protein samples.

Procedure for preparing muscle samples

1. Label each of the 4 sample buffer tubes, one for each type of tissue.

2. Cut a small bit of each tissue (size of half a pencil eraser) and put it into the corresponding tube.

3. Gently shake tubes and let samples sit for 5 minutes.

4. Label 4 empty microfuge tubes, one for each type of tissue.

5. Pipette 1/4 of the liquid (not the tissue!) from your sample tubes to the new tubes.

6. Incubate the tubes in the heat block at 95°C for 5 minutes.

7. The samples are ready to load into the gels. Be sure to keep track of which samples are loaded into
which wells.

Electrophoresis

1. When the agarose gel is hard, remove the stoppers.

2. Pour Tris-Glycine-SDS buffer over your gel so that is it completely covered plus a little more.

3. Draw a diagram of the gel including the wells. Label which protein samples you are putting into
each well because once the samples are loaded, you will not be able to determine which sample is which.

4. Load as much of each protein sample as you can into its well.

5. Run that gel!! Plug the electrodes into your electrophoresis apparatus (red to red, black to black),
being careful not to bump your gel too much. Plug the power source into an outlet and set the voltage to about 100 V.

6. Let the gel run until the dye migrates about 6-8 cm from the wells (about 20-30 minutes).

7. Turn off the power supply, disconnect the electrodes, and remove the top of the electrophoresis
apparatus.

8. Carefully remove the gel and place it in the staining tray.

9. Pour just enough Coomassie blue stain over the gel to cover it.

10. Cover with plastic wrap and stain for at least 30 minutes. [The gels can stain for longer, but the longer they stain, the longer they will take to destain.]

11. Remove stain and pour enough destain on the gel to cover it. Tuck a paper towel or some kleenex in the holder with the gel (it will soak up the stain).

12. Cover with plastic wrap and destain overnight.

13. Put the gel holder with the gel in it on the light box and view your gel. Draw a picture of your gel:

Analysis:

1. Compare the protein fingerprints from the different tissues. What can you conclude about what proteins each type of cell makes?
 
 
 

 
 
 
 
2. Which tissues had the most similar proteins? Which tissues had the most different proteins? Why do you think so?
 
 
 

 
 
 
3. Why would proteins from different types of cells look different? Why would they look the same?

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BIOTECH Project Department of Molecular and Cellular Biology The University of Arizona October 6, 1997 Last Modified March 5, 2002 Nadja Anderson, Ph.D. nadjal@email.arizona.edu http://biotech.biology.arizona.edu