EMSA Protocol
Jon Moore, Jan 2007
Steps
A)
B)
C)
D)
E)
F)
G)
H)
Pour the acrylamide gel.
End label the probe.
Purify the radiolabeled probes.
Set up the incubations.
Run the gel.
Drying the gel.
Exposing the gel.
Developing the film.
A) Pour the acrylamide gel.
1) Clean the plates, spacers and combs, first with diH20, then with Windex, then
with 70% EtOH. Paper towels are fine.
2) Set up the spacers (left, right, and bottom) and plates and clamp them with
super-large black paper clips.
3) The recipe for gel mix is:
9 mL 40% 29:1 acrylamide solution (leads to a 6% gel)
6 ml 5x TBE (leads to a 0.5x gel)
44.3 mL H20
600 ul 10% APS (I think I used half this at Millennium.)
(Don’t premix this ahead of time; over time these four ingredients
will partially polymerize without TEMED.)
60 ul TEMED (I think I used half this at Millennium.)
*** Don’t add the TEMED yet. ***
4) Mix this, being very careful since acrylamide is a potent neurotoxin. Pour 2-4
mls into a tube and add the proportional amount TEMED. Use most of this to
“seal the gel,” by pouring it into the top and rolling it around the sides and bottom.
Tilt the gel to leave this stuff on the bottom. Leave it alone until the remainder of
the 2-4 mLs polymerizes.
5) Now add the rest of the 60 uL of TEMED to the rest of the gel mix. Pour the
gel, being careful of air bubbles. Insert the 20-well square-tooth comb, being
careful to keep it straight and centered, and to not catch some of the mix in your
eye; wearing safety glasses is a good idea.
6) After the leftover gel mix has well polymerized, the gel is safe to handle.
(Keep in mind that no reaction, this one especially, will truly go to
completion; hence the polymerized acrylamide is much safer than
the unpolymerized stuff, but still be careful with it and don’t handle it
with bare hands. I believe that repeated exposure to polymerized
acrylamide might have been the reason for a tingling pain I had at
Millennium Pharmaceuticals. It could have been RSI as well.)
7) Wrap the comb end and put it in the cold room overnight. If stored for more
than overnight, add a little 0.5x TBE to the comb end to keep it from drying out
and wrap the other end as well.
8) Mix 1 L 0.25x TBE. Add 50 mL 5x TBE to a 1L flask. Invert with Parafilm
over the top, and place in the cold room.
B) End label the probe (primer pairs).
1) This assumes that they are annealed. I annealed mine at 10 pmol/ uL by
adding two 10 uL 100 pmol/ uL complementary primer stocks to 80 uL 10 mM
Tris, pH 8.0, 1 mM EDTA, and 62.5 mM NaCl, and then placing in the
thermocycler at 98 C for 2 min, and then a ramp to 4 C of -0.1 C / sec. You can
also use the heat block; heat it to between 95 C and 100 C, add your samples,
and turn off the heat block. An hour later they should be annealed at room
temperature.
2) This is a simple enzymatic reaction. For each reaction, mix 38 uL H20, 5 uL
10x T4 polynucleotide kinase buffer, and 1 uL T4 polynucleotide kinase. (I made
a master mix of this, and then added 44 uL to tubes.)
3) Then add 1 uL (10 pmol) of the annealed primers to each.
4) Now go the radioactive room and take all the proper precautions. Add 5 uL
(50 uCi) of gamma-P32 labeled ATP to each. Mix.
5) Incubate at 37 C for 30- 60 minutes; because of a couple casual comments
made, I have small reason to believe that more time is good. Place something
on or over the water bath so that people know that radioactivity is in there.
C) Purify the radiolabeled probe.
(This is a small modification of the Roche protocol.)
1) Find the columns in the radioactive fridge.
2) Remove the column from the storage container and gently invert it several
times to resuspend the medium.
3) Remove the top cap from the column; then remove the bottom tip. This
sequence is necessary to avoid creating a vacuum and uneven flow of the buffer.
Allow the buffer to drain by gravity (2-5 min) and discard.
4) Place the column in a collection tube, place this in a 15 mL snap-cap tube,
and centrifuge at 1100 x g for 2 minutes. (This is around 2,200 rpms in the
clinical ’fuge, which is, I think around 3.5 on the dial.) Discard the collection tube
and the eluted buffer.
5) Keeping the column in an upright position, very carefully apply the radioactive
DNA sample (up to 100 μl) to the center of the column bed. Avoid applying the
sample to the sides of the column; if this occurs, nucleotides flow around the
medium and are not retained. Overloading the column (volume >100 μl) also
results in nucleotides flowing through, contaminating the DNA sample.
6) Being careful to keep the column in an upright position, place the column in
the second collection tube. Maintaining the column in an upright position is very
important, especially after centrifugation. Tipping the column causes back-flow of
the DNA sample, resulting in reduced recovery.
7) Centrifuge for 4 minutes at 1100 x g in a snap-cap tube.
8) Save the eluate from the second collection tube. This contains your purified
DNA sample.
9) Add 150 uL T10E to the eluate to bring the total volume to 200 uL.
10) Discard the column into a designated radioactive waste container.
11) Clean up after yourself and check all those areas used in B & C with Geiger
counter.
D) Set up the incubations.
This part here is different for many different gel shifts. Below is
what I did on 1-8-07, but nearly all components could be tweaked.
Keep in mind 2 things: (1) You will nearly always want to run (a)
your labeled probe by itself (It frequently doesn’t run as a single
nice clean band.) to compare to your experimental sample, (b) your
labeled probe plus cell extract to see if it will shift some of the
sample by binding, and (c) your labeled probe, cell extract, and a
bucket of unlabeled probe to see if you can titrate off whatever is
shifting your probe. (2) Use buckets of dIdC to bind up the
proteins which bind DNA in a nonspecific way.
Some complexes form better at room temperature; others do better
on ice. I have no great wisdom on this front. I did all my initial
experiments on ice simply because it seemed the safer option
given how much effort one goes to keep nuclear extracts from
degrading.
1) Your total binding assays will be 20 uL. What you’ll need is
1 uL hot probe.
4 uL 5x binding buffer.
0.3 – 0.5 ug dIdC per ug nuclear extract (usually 1 ug/uL).
possible 2-5 ug nuclear extract.
Try to keep this volume well below 10 uL. Otherwise, the
nuclear extract buffer will play too great a role.
Possible cold competitor probe. A good initial trial is 200 fold
excess, which is 1 uL if you’ve followed the protocols above.
Water to 20 uL.
2) Thaw the nuclear extracts on ice.
3) Combine everything except the probes, adding the much more precious
nuclear extract last. Incubate on ice for 10-15 minutes.
4) Add the cold competitor probe, if using. Move to the hot room.
5) Now add the hot probe. Incubate on ice for 15 minutes.
6) While incubating, get the gel ready.
5x Binding buffer composition based on Ortiz, et al.
75 mM Hepes 7.9
400 mM NaCl
75 mM KCl
0.1 mM EDTA
5 mM DTT
5 mM PMSF
15% glycerol
E) Run the gel.
1) Take off the large paper clips and plastic wrap. Gently remove any loose
acrylamide from the top of the gel.
2) Add marks to the gel plate to help you to see the wells later, if necessary.
Gently and slowly pull out the comb. If vacuum is forming behind comb, it can
make the walls of the wells wiggly and not uniform. If this is occurring, slow
down and try gently wiggling the comb. (Do not be forceful! If you do, you run
the risk of pulling the two plates apart, and then all is lost.)
3) Put the gel into the apparatus with the notched plate away from you. Use the
four screw attachments to clamp it into place and press it against the gasket.
4) Add the 0.25x TBE to the gel apparatus both in the central compartment
which will cover the top of the gel and in the lower compartment. Make sure
there is plenty to cover the top and make good contact with the bottom. Note the
level of the TBE is at the top. (See step 6.)
5) Bring the power supply (near the western window in 202) into the cold room.
It should not stay there because the cold and condensation is not good for it.
6) After 10 minutes or so, check to see that the level of the TBE has not
changed. If it has, gasket has leaked, and try steps 3 and 4 again, or decide that
the gasket has degraded.
7) Get your samples and load your gel. The glycerol in the binding buffer should
facilitate the decent of the samples. Note that the samples are hard to see, and I
kneel to see them better, which is part of the reason I wear safety glasses while
doing this. You’ll see the decent of your samples as shadows against the
apparatus.
8) Load DNA running buffer so that you can keep track of how the gel is running.
If you have a free lane, add 2-3 uL there. If you don’t add 0.5-1 uL on top of one
of your probe alone lanes.
9) Plug in the gel and set the gel to run at 35 amps.
10) Monitor the gel. When the faster dye gets 80% of the way down the gel,
stop it. This will take about 1.5 hours, though the gel speeds up as it runs, so be
careful.
I think this speeding up is due to the constant amps setting and the
difference between the gel’s buffer concentration and the running
buffer concentration. Basically, the voltage goes from 250-300 V at
the beginning to 500-550 V after an hour and a half, and one would
expect that as the gel composition changes, then the resistance
would increase by about a factor of 2, leading to a voltage change
of a similar factor. I’m inclined to one day try running the gel at
400-500 V to see what happens. What I am not sure of is what
concentration of TBE I should use. I tend to think it should be 0.25x
all around.
11) Stop the gel.
F) Drying the gel.
1) Cut a piece of Whatman paper an appropriate size. It should be bigger than
the gel yet small enough to fit in one of the gel cassettes.
2) Remove the gel from the apparatus. Start with the bottom screws, since if
you are fast enough, you can loosen these before buffer covers the screws.
3) Pull the plates apart by slowly pulling up on one of the spacers hanging out.
Be sure that the gel remains relatively flat. If it seems to be sticking to the upper
plate more than the lower one, flip it over so that the gel doesn’t need to fall.
Make any adjustments you might need to make to make the gel flat. If something
truly awful happens in terms of bending and folding, a squirt bottle with water can
be useful for moving the gel around without picking it up and risking breaking it.
4) Press the Whatman paper on top of the gel. When you remove it, the gel
should come with it.
5) Take the gel to the gel dryer near the western window of 202. Check the
agroscopic compound which runs between the dryer and the vacuum; replace it,
if need be.
6) Open the dryer; pull back the flexible plastic sheet. Put the gel on the dryer
paper-side down. Place Saran wrap over the gel, and now pull the flexible plastic
sheet over the whole thing.
7) Turn on the vacuum. You will likely hear a hissing noise, which means the
sheet and the base are not forming a seal. Make sure the sheet is straight, and
lightly run your finger around the edge to help form a seal. Likely, there will be
one spot which still doesn’t seal; gently tug and press so that the sheet is flat and
that a seal is made.
8) Close the lid and push the Start/Stop button. This activates the heat for the
gel dryer; the timer is generally set for the heat to go off in 2 hours, but check it.
9) After two hours, your gel will usually be toasty-warm and dry. Remove it, turn
off the vacuum, check the area for radioactive contamination, and discard the
Saran Wrap.
10) Write something on the Whatman paper so that you can distinguish this gel
from the next ten you’ll be running.
G) Exposing the gel.
1) Find an approximately 9” by 11” hinged gel cassette, and a box of Kodak
BioMax XAR Film, 8” by 10”. Take these and your gel to the dark room.
2) Pull the hinge to open the cassette. The screen is the white part, and black
part is the bottom. Put the gel paper-side down on the bottom of the cassette,
preferably near the bottom-right corner.
3) Turn out the light. Now read the rest of the protocol. 
4) Open the box of film and remove one piece of film; they tend to stick together.
Crimp one corner of the gel for orientation. Place the film covering the gel with
the crimped part toward the top-left. Close the cassette; if the film or gel moves,
you may not be able to close it; if so readjust them.
Note that messing with the film too much can leave fingerprint-like
marks. I’ve also noticed things which look like static discharge. Try
to keep this to a minimum.
5) Return the inner envelope of film to the box open-side down. Close the box,
and turn on the light.
6) If multiple gels cassettes are in use, label the cassette with tape.
7) Put the cassette at -80 C for 1 hour to 1 week. Overnight is a good starting
try.
H) Developing the film.
1) Remove the cassette from the freezer and let it return to room temperature,
about 15 minutes.
2) Take it to the door at the base of the ramp in the basement of Kerckhoff.
Keep going through that set of rooms until you find the darkroom.
3) Usually the developer is on. If not, follow the directions on the wall.
4) Close the door and open your cassette. Place the film on the tray on the right
of the developer, snug to rest of the developer. Press the button in front of the
tray and the developer should start slowly pulling in your film.
5) The machine will beep when the film is safely inside the machine. You can
then feed another film to the machine now if you have one. Otherwise, during
the two minutes it takes to develop, open the door and fill out the log on top of
the machine.
6) The machine will drop your developed film into the bin on the left. If
developing more than one film, make sure to note the order they come out.
If you have two films stick together in step G) and you feed them
into the developer together, it won’t work well, so be careful.
7) Write the important information on the gel film.