BIO 330 Cell Biology
Laboratory
Spring 2011
Western Blotting: Detection of Specific Proteins in Biological Samples
The study of proteins in biological settings allows us to understand the role these proteins play in real
life. Unfortunately, biological settings are enormously complex, so it can be difficult to isolate and purify
individual proteins. Lucky for us, the vertebrate immune system has provided a tool with which we can
detect even the rarest of proteins in a soup of proteins, DNA, carbohydrates, and lipids. These tools are
called antibodies, and you have already been introduced to them during the ELISA lab.
Antibodies are used in a variety of assays to determine whether particular proteins are present. These
range from ELISAs, which are used for large-scale detection of proteins in many different samples, to
RIAs (radioimmunoassays), which are similar to ELISAs but use radiation as a marker, to fluorescenceassisted cell sorting, to western blotting. Western blotting has the advantage over all these other types
of assays of giving the investigator information about the size of a protein in addition to simply its
presence. Because of this, western blotting is considered more specific and precise than the other
methods. Frequently, antibodies are validated using western blotting before being used in other
applications to ensure they are specific.
Western blotting involves several major steps. First is the separation of proteins based on size using
polyacrylamide gel electrophoresis. Next, the proteins must be transferred to a sturdier substrate, a
nitrocellulose or PVDF membrane. Third, the membrane is probed with a primary antibody, followed by
secondary antibody, followed by detection in order to determine which samples contain the protein of
interest, and how large the protein of interest is.
In this activity, we will be using specific antibodies to test for the presence of a hormone called
adiponectin in pig plasma. The first week, we will run pig plasma in several wells of a polyacrylamide
gel, then transfer the proteins to two different membranes – one nitrocellulose, and one PVDF. These
membranes will be saved until next week. At that time – at the Portland VA Medical Center – each
membrane will be subjected to a similar, but varying, protocol in order to optimize the conditions for
our particular antibody.
Protocol
1. Prepare your samples. Each student should mix 50 uL of pig plasma with 50 uL of 2X Laemmli loading
buffer in a medium microcentrifuge tube. This buffer contains SDS (sodium dodecyl sulfate), a
detergent which will unfold proteins and coat them with a negative charge; as well as DTT
(dithiothreitol), a reducing agent which will break apart disulfide bonds, completely denaturing proteins.
Using this buffer means we are running our gel under denaturing conditions. After mixing your samples,
close the lid securely, and boil for ~5 min by placing in the 100°C heat block (it’s OK if you don’t actually
see it boiling). Then place on ice.
2. The class will be sharing two gels, which will be run on one apparatus. Set these up according to Dr.
DuPriest’s instructions. Each student should then pipette 20 uL of pig plasma into 3 wells according to
Dr. DuPriest’s instructions. One lane on each gel (preferably in the middle) should have 10 uL of a ladder
instead of pig plasma.
BIO 330 Cell Biology
Laboratory
Spring 2011
3. Place the lid on the gel rig, and set the power supply to 120 V. Run for ~30-45 minutes, until the dye
front is at or near the end of the gel.
4. During the last 10-15 minutes of the gel running, prepare your transfer. Set up two flat trays, and fill
one with 100% methanol; fill the other with transfer buffer, which should be pre-chilled. Make sure the
following materials are ready: cassette (gray and black); one small rectangle of each type of membrane;
4 square of blotting paper; razor blade; forceps. NOTE: USE GLOVES WHEN HANDLING METHANOL OR
TRANSFER BUFFER (WHICH CONTAINS METHANOL), AS IT IS POISONOUS.
5. After the gel is finished running, turn off the power, and gently remove the gels from the gel rig. Lay
the gels flat on the table, and open the cassette carefully according to Dr. DuPriest’s instructions. Using
a razor blade, cut off wells, the sides of the gel, and the bottom of the gel below the dye front. Carefully
pick up the gel and soak it in transfer buffer for 10 minutes. At the same time, cut your membrane so it
is just a little larger than the gel. NOTE: DO NOT TOUCH THE MEMBRANES WITH YOUR FINGERS. USE
FORCEPS, AND HANDLE ONLY BY THE EDGES/CORNERS. Presoak membranes in flat trays according to
the following instructions. For nitrocellulose: soak in methanol for 5 minutes, then soak in transfer
buffer for 5 minutes. For PVDF: soak in transfer buffer for 10 minutes. During this time, fill a larger tub
about ½ full with transfer buffer – do this last so it stays chilled. Also, while you are waiting, pour out
the running buffer into the sink, and rinse out the gel rig…you will use it for your transfer.
6. Assemble your “transfer sandwich.” To do this, place your transfer cassette, gray side down with the
black side up and resting against the side, in the tub with transfer buffer. Place a piece of blotting paper
on the gray part of the cassette. Place your gel FACE DOWN on the blotter paper. Carefully center your
membrane over the gel, and lightly roll over the membrane using a piece of plastic pipette to remove air
bubbles from between the gel and membrane. Then place another piece of blotting paper over the gel.
Carefully close the cassette, keeping everything submerged as much as possible, and trying to keep the
gel and membrane from sliding. If the cassette is too loose, you can add 1-2 more pieces of blotting
paper to make the sandwich thicker. Do not make it too thick!
7. Fill the gel rig about halfway with ice-cold transfer buffer, and place the ice pack in place. Take the
whole transfer sandwich and slide it into place in the gel rig, with the gray side facing the red electrode,
and the black side facing the black electrode. The second group should repeat the sandwich assembly
and load the cassette into the transfer apparatus.
8. Place the lid on the chamber, connect electrodes to the power supply. Set the power supply for 4V
(no, this is not a typo…NOT 40V). The transfer will run very slowly overnight. If power cords will reach,
we may place this in the refrigerator.
9. In the morning, Dr. DuPriest will disassemble the transfer sandwich, and cut each membrane into 2
sections. These will be saved in Tris-buffered saline (TBS) until next week’s lab.
10. The following week, the membranes will be placed by Dr. DuPriest in one of two different blocking
solutions prior to your arrival. This is to determine which blocking solution works best for the two
different kinds of membranes for this particular antibody. The first blocking solution is 3% BSA (bovine
BIO 330 Cell Biology
Laboratory
Spring 2011
serum albumin); the other blocking solution is 5% nonfat dry milk. Both blocking solutions contain the
given component dissolved in TBST (TBS plus 0.01% Tween-20, a mild detergent), which will also serve
as our wash solution in the following steps. Your membrane will be incubated in its appropriate
blocking solution at room temperature for 60minutes.
11. Near the end of the 60 minutes, prepare your primary antibody solution in a 15-mL conical tube.
Label the tube, and save it! This should be a 1:1000 dilution of the primary antibody (mouse anti-human
adiponectin) in your appropriate blocking solution.
12. After the 60 minute blocking period, pour the blocking solution back into its appropriate
container…do not mix the two solutions! Make sure you get every last drop possible, without losing
your membrane. Pour the primary antibody solution onto your membrane, place the lid on the tray,
and incubate on the rocking platform at room temperature for 60 minutes.
13. After the 60 minute primary antibody incubation, pour the antibody back into the same tube you
used to prepare the solution. Place on ice. Pour enough TBST wash solution in the tray to generously
cover the membrane. Put the lid on and incubate on the rocking platform for 5 minutes. Pour the
solution into the sink, and repeat two additional times (total of 3 washes).
14. Pour more blocking solution on the membrane, and incubate for 30 minutes as described above.
Near the end, prepare your secondary antibody (goat anti-mouse HRP conjugated)) solution as
described for your primary antibody. This time, your antibody should be prepared at a 1:5000 dilution.
15. Pour off blocking solution into the sink (NOT back into the blocking solution container this time).
Pour your secondary antibody solution onto the membrane and incubate for 60 min as described.
16. Pour off secondary antibody solution into the sink and wash with TBST 3 times as described above.
17. During the last wash, prepare your detection solution according to Dr. DuPriest’s instructions. After
the last wash, pour off your wash solution, then pour the detection solution onto the membrane and
incubate for 1-5 minutes, according to instructions. During this time, prepare your film cassette. You
should cut a piece of plastic transparency in half, then trim the edges so it will fit in the cassette. After
the incubation, grasp the corner of the membrane with forceps, let it drip for a few seconds, then
carefully blot the edge on a paper towel to remove excess solution. Place face up on a transparency,
then place the other transparency on top, and use a Kimwipe to remove any air large air bubbles. Tape
the transparency so it is flush with the upper left corner (with hinge of the cassette at the top). Take the
cassette and box of film to the dark room.
18. Dr. DuPriest will demonstrate how to expose and develop film with one piece of film per cassette,
then students will try with a different exposure length. Once satisfied with results, take everything back
to the lab. Clean everything up according to Dr. DuPriest’s instructions.
BIO 330 Cell Biology
Laboratory
Spring 2011
Conclusion/Discussion
Describe what your results mean. For this lab, we used two kinds of membrane and two kinds of
blocking buffer. Which combination worked best? Should the primary or secondary antibody
concentration be adjusted for future work, and if so, what dilution of each would you suggest? Explain
what we can tell about adiponectin in normal pigs vs. low-protein pigs, if the results are clear. If this
were a real experiment, what else would we have to do to make sure our data are high quality? What
other experiment could I do to know for sure if we are detecting adiponectin and not some other
protein?