COMPARING PROTEIN PROFILES FROM DIFFERENT TISSUES
Sample Preparation
1. Label 1.7 mL tubes with 500 l of Laemmli Sample Buffer. Label the tubes so that you know
which sample is in which tube.
2. Place a piece of your tissue sample that weighs 0.1 grams, into the Laemmli Sample Buffer. Make
sure the tissue sample is completely submerged.
3. Let tissue sample set at room temperature for 5 minutes. If you feel like you have a lot of solid
chunks, centrifuge on high for one minute
4. Transfer approximately ~200 l of sample liquid to a newly labeled 0.65 ml tube. Make sure to not
transfer any solid material.
5. Place the 0.65 ml tubes with the liquid extract into 95°C heat block for 5 minutes. This will
completely denature the extracted proteins.
Proteins are now extracted and denatured and ready for gel electrophoresis. Proteins can be stored in 20°C until ready to run gel, however, they will need to be denatured again prior to running on gel.
SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Vertical electrophoresis for proteins with acrylamide gel is a bit trickier, more complex and more
hazardous than horizontal electrophoresis done with DNA and agarose gels.
1. If samples are frozen, take them out of the freezer and thaw. Place liquid protein samples into a
95°C heat block for five minutes to denature the proteins so they can easily move through the gel.
2. Inside the gel box is the gel cassette. Take the gel cassette out of the gel box
3. On one side of the gel cassette is a
buffer dam. If you are running one gel
the gel cassette leave the dam in place.
you are running two gels in the
cassette, remove the dam. (DO NOT
THROW THE DAM AWAY!)
in
If
4. Put on gloves and get a package that holds your gel.
The gel incorporates a stacking gel that has been dyed red.
In addition, there is no comb to remove as the top of the teeth are fixed in place. This not only
prevents the teeth from being accidentally torn off during comb removal but that all of the wells are
uniform, with the teeth remaining vertical. This prevents sample cross contamination/inability to
load desired volume. The teeth also protrude above the lower front plate of the gel cassette
preventing sample flow over into adjacent wells.
5. Open the sides of the gel cassette. Place the gel in the gel cassette so you see the numbers written
correctly from the outside of the gel cassette (This is critical so ask if you are unsure). Place the
second gel in the same way or make sure the dam is placed correctly in the gel cassette. Close the
sides of the gel cassette
6. Place the gel cassette into the gel box. Carefully remove the comb (The gel is very fragile), and
load the gel with protein samples (10l). Don’t forget to record which sample is in which lane.
Don’t forget to load a protein standard to monitor movement of proteins (5l).
7. Here is another critical step. Fill the inside of the gel cassette to the top with Tris Trycine/SDS
buffer. Outside the gel cassette, fill the gel box up to appropriate line.
8. Plug the gel box into the power supply. Set the power supply to 200 volts and start the run. If you
protein standards are prestained, you can see the protein standard to know when to stop the gel.
The smallest molecule of protein standard should about 1.5 centimeters from the bottom of the gel
(about 20-30 minutes). If the standards are not prestained, then monitor the progression of the gel
with the dye front and stop the gel when the dye front runs off the gel.
9. To stop the running of the gel, turn off the power supply and remove the gel from the gel cassette.
Open the gel casing and carefully (gel is thin and delicate) separate the two halves of the casing.
The gel will stay on one side or the other of the casing and usually the bottom will curl up, that is a
good place to gently pull gel up off the plate and place into the staining tray. BE CAREFUL
WITH GEL, IT IS VERY EASY TO TEAR!
10. Pour enough Coomassie blue stain to cover the gel. This is a powerful stain, be careful not to
splash it on self or clothes, as it will stain any and all proteins it touches (including yours!).
11. Stain the gel, shaking gently for 30 minutes or longer. After 30
minutes, recycle the stain by pouring it back into the bottle.
12. Replace the stain with destain (which will smell like vinegar) –
completely covering the gel. Cover the tray and destain
overnight. Place two kimwipe balls into the destain to absorb the
destain and allow more destaining to occur.
13. The next day, place the gel on a light box for examination.
FINAL ANALYSIS
Remember – The distance the protein band has traveled is related to the size of the protein. Also, how
dark the band is tells the concentration of the protein– the darker the band, the more of that protein
there is.
What similarities and differences did you see between the protein profiles of the different samples? Is
this what you expected? Do similar tissues show more relatedness to each other (based on the # of
similar sized proteins)? Why are the protein profiles different for these tissues if the DNA in the
organism is the same in all of the cells?