COMPARING PROTEIN PROFILES FROM DIFFERENT TISSUES
Sample Preparation
1. Label 1.7 mL tubes with 500 l of Laemmli Sample Buffer (minus bromophenol blue, usually
this solution is blue, you will later use the blue version of Laemmli’s), so that you know which
sample is in which tube.
2. Add a piece of your tissue about the size of half of an eraser head, about 2-3 mm cubed. Make
sure the tissue is completely submerged in the Laemmli Sample Buffer.
3. Let 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 tissue.
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.
6. 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.
Determining Protein Concentration
Prepare Samples: You will be determining the protein concentration of your samples in groups of 48 students using protein standards of known concentrations
1. Label cuvettes and lids with your sample or your name.
2. For the protein standards, label 7 cuvettes and lids with the known concentrations of the protein
standards (see the data table).
3. Label an 8th cuvette and lid “Z” for Zero.
4. Add 125 l of prepared Reagent A into each cuvette.
5. Pipette 25 l of the your protein sample or the known protein standards into labeled cuvettes.
For the cuvette labeled “Zero” add 25 l of clear Laemmli’s Sample Buffer.
6. Add 1 ml reagent B into each cuvette, cap it, and vortex or invert several times immediately.
7. Wait 15 minutes and read absorbance at wavelength of 680 nm.
Collecting data
8. Wipe off the clear side of the Zero cuvette using a Kimwipe.
9. Place the Zero cuvette in the Colorimeter. Line up the clear sides with the arrow. Push the Zero
button and remove cuvette when you see 0.00. You should only have to Zero one time.
10. With the other cuvettes, wipe off the sides using a Kimwipe and place them in the Colorimeter.
Line up the clear sides with the arrow. Push the Test button and record the absorbance values.
COMPARING PROTEIN CONCENTRATIONS & PROFILES FROM DIFFERENT SOURCES
Determining protein concentration of samples.
Standards:
Protein concentration
(mg/ml)
0.0 (blank)
0.125
0.25
0.50
0.75
1.00
1.50
2.00
Absorption
0
Use Excel to find out the equation for the linear best fit line and write it below (these instructions
are for Excel in Windows 7, yours may differ):
1. Open an Excel spread sheet containing a column for protein standard concentrations, and
an additional column with their corresponding absorbances (as shown above).
2. Highlight the values, click on insert, and choose the scatter plot with no line function.
3. Go to chart layouts, choose trendline. In order to see the equation, go into trendline options
and click “display equation on chart”.
4. Determine protein concentrations of your insect samples using displayed equation.
Tissue Samples: Raw data
Sample name
Absorption
Tissue Samples: Processed data
Sample name
Absorption
Protein
concentration
(mg/ml)
Analyzing data and calculating dilutions for SDS-PAGE.
You will want to load an equal amount of protein for your protein gel. This is most easily achieved
if you start with all of your samples at the same concentration. If your concentrations are > 1 mg/ml
you can dilute your samples to be a standard 1 mg/ml of total protein. Below is an example of how
to calculate your dilution. If your above concentration is < 1 mg/ml, then you will need to add more
volume of your sample to the gel so that your sample has the same amount of total protein to the
other samples in the gel (more on that below).
Calculation for sample dilution to 1 mg/ml
For each sample, calculate how much of the sample to add to Laemmli Sample Buffer (with
bromophenol blue, make sure this solution is blue) to make 0.1 ml (= 100 l) volume. Example: if
the measured concentration of your sample was 3 mg/ml, use the following formula to make a final
volume of 0.1 ml of 1 mg/ml protein:
(final concentration) X (final volume) = (sample concentration ) X (sample volume)
(final concentration) X (final volume)
--------------------------------------------- = sample volume
Sample concentration
(1 mg/ml) (0.1 ml) = (3 mg/ml) (x)
(1 mg/ml) (0.1 ml)
----------------------- = (x)
(3 mg/ml)
x = 0.0333 ml = 33.3 l
Volume of Laemmli Sample Buffer needed = (100 l – sample volume)
Volume of Laemmli Sample Buffer needed = (100l – 33.3l) = 66.7l Laemmli Sample Buffer for
a final concentration of 1 mg/ml.
Sample name
Final protein
concentration
Volume of
sample needed
Volume of
Laemmli
needed
1 mg/ml
1 mg/ml
1 mg/ml
1 mg/ml
SDS Page electrophoresis
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
in the gel cassette leave the dam in
place. If you are running two gels in
the cassette, remove the dam. (DO
NOT THROW THE DAM AWAY!)
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 (15l if your concentration is at 1 mg/ml)). Don’t forget to
record which sample is in which lane. Don’t forget to load a protein standard to monitor
movement of proteins (10l). If your sample protein concentration is < 1 mg/ml, then you will
need to calculate how much sample you need to add to be equivalent to 15l of 1 mg/ml protein.
For example, if you had 0.5 mg/ml concentration you would need to add:
(15l)(1mg/ml) = (x)( 0.5mg/ml)
x=(15l)(1mg/ml)/0.5mg/ml = 30l
However most of you should have >1mg/ml protein concentration and will not need to do this step.
7. Here is another critical step. Fill the inside of the gel cassette to the top with Tris
glycine/SDS buffer. Outside the gel cassette, fill the gel box up to appropriate line (2 gel or 4
gel).
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, then watch 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. 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.
Destain for about 30 minutes. Pour the blue destain into the destain waste container and add
fresh destain into the tray. Cover the tray and destain overnight.
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?