Gel Electrophoresis Analysis of Proteins
Background Info: Different tissues of the body perform different functions. These tissues are made of
proteins which can be identified. But what do proteins help with? Do similar proteins help with similar
functions in different organs or do similar proteins not relate at all to the function of the organ/structure.
Today you will be taking samples of different tissues and isolating the different proteins using gel
electrophoresis. There will be two different sets of tests that will be run. One group will run tests for heart,
skeletal muscle, stomach, intestine, esophagus, and kidney. The second group will run tests for a salivary
gland, thymus gland, teste or ovary, liver, diaphragm, and lung. After the materials have been placed in the
gel and run this will create a 'protein fingerprint' of different types of proteins. You will then compare the
information from your group of structures to the other group’s structures to identify which ones have common
proteins and which ones are most dissimilar.
Materials/Equipment Needed
For the class:
• hot water bath (95°C)
• thermometer
• 3% agarose in Tris-Glycine buffer
• BenchMark Protein Ladder
• Tris-Glycine- Sodium Dodecyl Sulfate (SDS) buffer
• Coomassie blue stain
• Destain
• Plastic wrap
For each student group:
• horizontal gel electrophoresis
apparatus, electrodes, power supply
• micropipette with micropipette tips
• 6 microfuge buffer tubes
• 6 empty microfuge tubes
• staining tray
• paper towel or kleenex
• different tissue
Procedure for preparing tissue samples and electrophoresis
1. Label each of the 6 sample buffer tubes, one for each type of tissue.
2. Pipette in 30ul of the Tris-Glycine-Sodium Dodecyl Sulfate (SDS) buffer into each tube.
3. Cut a small bit of each tissue (size of half a pencil eraser), squeeze with your fingers to break it
apart, and put it into the correctly marked tube.
4. Vortex each tube for 3-5 seconds and let the samples sit for 10 minutes. During the 10 minutes
vortex the sample every 2 minutes.
5. During the 10 minute wait time, label 6 empty microfuge tubes, one for each type of tissue.
6. Then add enough Tris-Glycine SDS buffer over your gel which is in your electrophoresis
chamber so that it completely covers the gel. Pour the buffer first into both ends of the
chamber and then continue adding more until it barely covers the top of the gel.
7. After the 10 minute wait time has passed, pipette 15ul of the liquid (not the tissue!) from your
sample tubes to the new tubes.
8. Heat the tubes in hot water (95°C ) for 5 minutes. This will denature the proteins so they will
go from a quaternary structure to a primary structure. A hole may have to be punctured in the
lid of each sample to reduce pressured build up due to the heating.
9. After the 5 minutes of heating has elapsed, vortex each microfuge tube.
10. Micropipette 10 uL each sample into a respective well gels. The first well should have the
BenchMark™ Protein Ladder pipetted into it. Be sure to keep track of which samples are
loaded into each well. Label this on the diagram below.
11. After the samples are in each well place the lids in the correct position on top of the chamber. Red
should connect to red and black should connect to black. Then plug the wires into the power supply
with the red wire going to the + and the black wire going to the -.
12. Next plug in the power supply into an outlet with the voltage being set at 100 V.
13. The dye should migrate about 6-8 cm from the wells (negative to positive charge) which will take
about 20-30 minutes. You should let your instructor know when you think it has migrated this distance
so they can verify it.
14. After the gel has run the correct distance, turn off the power supply, disconnect the wires from the
power supply, and remove the lid from the electrophoresis chamber.
15. Carefully remove the gel from the frame it is sitting in and place it in the staining tray.
16. Pour just enough Coomassie blue stain to cover the gel. This will stain the proteins.
17. Cover the staining tray with plastic wrap and let this sit for at least 30 minutes. Your instructor will
help to determine if they need to sit longer or if they are ready to be destained.
18. Slide your gel out of the tray and place it in another clean empty tray. Pour enough destain on the gel
to cover it. If the blue coloration comes off and floats to the top take some kleenex and place it on the
surface to soak up the stain.
19. Cover the tray with plastic wrap and destain overnight.
20. The next day put the gel on the light box and view your gel. Draw a picture based upon the migration
of the proteins in your gel below:
Analysis - Post Lab Questions
1. What was the general shape of the proteins before putting them in the 95°C water? What was the shape
after you put them in the 95°C water? Why couldn’t you simply eliminate adding the proteins to the 95°C
water step?
2. Do all cells produce the same proteins? Explain why or why not.
3. Are there any proteins which are made by all the tissues? If so which ones. If not are there groups of
tissues which have the same proteins in them, such as muscle? What function is accomplished by all cells
that the same protein would be needed for?
4. Are there any proteins made by only 2 or 3 organs? What do you feel would be the reason for these
organs making those proteins? Does it relate to their function?
5. If you took other cells from the same organ but different location would the proteins found in them be
the same or different? As an example if you took a tissue sample from beginning of the small intestine as
opposed to near the end, would you expect to have the same proteins found. Explain why or why not.
6. In terms of complexity do organs that perform many functions have the most proteins associated with
them? To help with this list the organ/structure and how many proteins are found next to it.