EMSA PROTOCOLS FIR 2010

Experiment 1: Electrophoretic Mobility Shift Assay (EMSA) to Evaluate Effects of

Hormone Treatment on Transcription Factor Binding to the cAMP Response Element

Overview:

1. The Leydig cell line MA-10 will be grown to approximately 95% confluency on four 150 mm culture
dishes. Prior to the start of the experiment, the TAs will have added four different treatments to the
cells and each group of 4 students will prepare extracts from a single150mm plate of MA-10 cells
with one of the treatments.

2. The treatment groups are as follows:

Group A = no treatment control (vehicle only)

Group B = + 1mM cAMP
Group C = + 10-8M DHT
Group D = + 10-8M DHT + 1mM cAMP

Results will be compared between groups. Also, Emily and Valentine will prepare and analyze a second no-
treatment control – DHT carrier.

Notes:

1. 8-bromo-cAMP is short for 8-bromoadenosine 3':5'-cyclic monophosphate; a long-acting, cell

permeable derivative of cyclic AMP that is resistant to degradation by phosphodiesterase

2. For treatments, media is removed from the cells and replaced with serum-free media containing
1mM 8-Bromo cAMP (300ul of a 100mM 8-Bromo cAMP [MW 8-bromo-cAMP = 430.1g/mol. 1ml
of a 100mM solution = 43.01 mg in 1ml serum-free DMEM/F12 media or sterile H2O] stock
solution into 30ml of serum-free RPMI-1640 media). Untreated cells receive fresh serum-free
media with the same volume of water/ media added for the cAMP treatment. Cells are incubated
for 2hr at 37oC.

3. 5α-dihydrotestosterone, DHT, is a metabolite of testosterone with greater androgen activity than

testosterone itself. Cells are first cultured in DMEM (without serum) and treated with 10-8M DHT
for 16-24 hours prior to harvest.
a. Treat cells as follows:
1. Dilute a 10-2M stock solution of DHT into DMEM to make a final concentration of
10-4M. Using a 1.5 ml microcentrifuge tubes make a 10-4M dilution of DHT as
follows: 990 μL of DMEM plus 10 μL of 10-2 M DHT stock
2. Remove the culture media from one 150mm plate of MA-10 cells and replace it
with 30mL DMEM containing 10-8M DHT. To make 30mL of media containing
10-8M DHT, add 3 μL of 10-4M DHT and mix.
3. Return the cells to the incubator and culture them with hormone for an additional
16-24-hours.

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 EMSA PROTOCOLS FIR 2010

Preparation of Nuclear Extracts

All procedures, supplies, and solutions should be at 4oC

The method for preparation of nuclear extracts is a modified version of that originally published by Dignam in 1983
(Dignam JD, Martin PL, Shastry BS, Roeder RG. Eukaryotic gene transcription with purified components. Methods
Enzymol. 1983;101:582-98). In this procedure, cells are harvested, washed, and resuspended in a hypotonic
buffer, which causes the cells to swell. The swollen cells are lysed by homogenization (using a hand-held Dounce
homogenizer) and the nuclei pelleted by centrifugation. The supernatant fraction (cytoplasm) is then decanted
and the nuclear pellet is resuspended in a moderate salt buffer and incubated 60 minutes to extract the nuclear
proteins. A second centrifugation step pellets the nuclear membranes and the resulting nuclear proteins are left in
the supernatant, which is used in the binding experiment.

*Note, all indicated volumes are for a single 150mm dish.

1. Turn on refrigerated centrifuge and set temperature to 4oC.

2. Make HEGD and HEGDK solutions. All solution recipes are on a separate sheet. Note,
these solutions must be made fresh.

HEGD is a hypotonic buffer that causes the cells to swell, thus facilitating disruption of the
cytoplasmic membrane [all indicated volumes are for a single 150 mm plate]

3. Remove media from one 150mm plate of MA-10 cells and wash the cells with 5ml HEGD
(plus Roche complete protease inhibitors) by gently swirling the solution to rinse the cells,
followed by its removal by aspiration.

4. Add 1.0 ml HEGD (plus protease inhibitors) per plate, dislodge cells using a cell
scraper and pipette the cell suspension into an ice-cold 5ml Dounce
homogenizer. Repeat with an additional 0.5ml HEGD, to maximize the cell yield.

5. Disrupt the cells using 30 strokes with a size “B” pestle. See figure

6. Transfer lysate to a cold 1.5ml microcentrifuge tube.

7. Centrifuge for 1 minute (max RPM in a microcentrifuge @ 4oC) and remove

supernatant (cytoplasmic fraction) by aspiration. Be careful not to aspirate any
of the pellet.

8. Estimate the volume of your pellet (contains the nuclei and should be about 100-120ul) and
resuspend it in HEGDK (extraction buffer) using a volume ratio of 1:1 (pellet to HEGDK).

9. “Stir” with pipette tip and vortex to mix (solution will become very “gooey”).

10. Extract proteins by incubating on ice for 1 hour.

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 EMSA PROTOCOLS FIR 2010
11. Centrifuge at 4oC and 45K rpm for 6 minutes using a TLA 100.4 rotor (85K x g avg.) fitted
with adaptors for microcentrifuge tubes.

12. Carefully remove the supernatant and place it into a clean 1.5ml microcentrifuge tube.
Place 10-15ul into a 200ul PCR tube for protein analysis and divide the rest into PCR tubes
as 30ul aliquots and store at -80oC. This is the nuclear extract (the pellet is the nuclear
matrix). Yield is approximately 150ul or 5, 30ul tubes.

13. Determine the protein concentration, using the protein assay described below.

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 EMSA PROTOCOLS FIR 2010

Protein Concentration Determination

1. Obtain one tube of frozen nuclear extract and let thaw at room temperature. Mix thoroughly
before pipetting the sample for protein quantification.

2. Prepare samples for standard curve.

a. Using the table below, calculate the amount of water and BSA stock solution (2ug/ul)
required for 40ul of each indicated standard concentration.

Vol. Vol. H20 Standard

Sample BSA stock* [q.s. to 40ul] concentration
(ul) (ul) (ug/ul)
0 (Blank) 0 40 0
1 0.05
2 0.1
3 0.15
4 0.3
5 0.6
6 0.8
7 1

*BSA protein stock solution is 2ug/ul.

b. Prepare each standard, mix, and aliquot 10ul, in triplicate, into three wells of a 96-well
clear bottom black microplate (i.e. 10ul of the 1ug/ul standard into A1, A2, A3).

3. Thaw one tube of your frozen nuclear extract and mix thoroughly.

4. Pipette the sample: 1µl, 3µl and 5µl in triplicate into 9 wells (i.e. 3 wells each) of the 96-well
plate. The total volume in the each well before the addition of the protein dye reagent should
be 10µl; add the required volume of ddH2O to obtain 10µl total volume in each of the 9 wells.

5. Dilute the 5X protein dye reagent (BioRad) to 1X with ddH2O (i.e., add 4 volumes of water to
1 volume of dye reagent).

6. Add 200uL to each well. Mix thoroughly using a microplate mixer for about 5 sec.

7. Check the samples to make sure there are no bubbles, which may cause inconsistent
absorbance readings.

8. Incubate at room temperature for 5 min.

9. Read the absorbance at 595 nm in 96 well plate reader

10. Prepare a standard curve and use it to determine the protein concentration of your nuclear
extract.
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 EMSA PROTOCOLS FIR 2010

In Vitro Transcription/Translation
1. Set a water bath or heat block to 30oC (make sure temperature has stabilized before thawing
reagents).

2. Note that all reaction components should be kept on ice, while working with them at your
bench. Also, since the intermediate step is the generation of mRNA for translation, the
samples must be maintained in an RNase-free environment or the generated transcripts will
be degraded and lower your yield. See precautions for RNase in the supplementary
material.

3. Retrieve the TNT reaction kit from the freezer (–80°C, 3rd floor) and immediately place the
RNA polymerase in a bench-top cooler that has been stored in the freezer (-20oC).

Note, when using enzymes, precautions need to be taken to prevent

temperature fluctuations, as this causes loss of enzyme activity.
That is why we are using bench-top coolers, which maintain freezer
temperatures for extended periods at room temperature.
Enzymes are placed in the cooler directly from the freezer and kept
in the cooler while using them at the bench.

4. Rapidly thaw the reticulocyte lysate by rubbing the tube between your hands and then place
it immediately on ice. The buffer and amino acid mixtures can be thawed at room
temperature and then stored on ice.

5. While one student is thawing the lysate, another can add the water and reaction buffer to two
0.5 ml microcentrifuge tubes (labeled to indicate +CREB-cDNA and Uncharged or no-DNA).
Once the reticulocyte lysate thaws, add it to each tube and immediately return remaining
lysate to the -80oC.

6. Add the rest of the reaction components (in the order given) for the CREB and Uncharged
TNT reactions. After all components are added, gently mix by slowly pipetting up and down
(do not vortex). If necessary, briefly centrifuge (tap spin) to collect the reaction mixture into
the bottom of the tube.

TNT REACTION
Component CREB Rxn Uncharged Rxn
Volume (uL) Volume (uL)
Nuclease-Free Water (q.s. to 50ul) 19.0 20.0
Reaction Buffer 2.0 2.0
Rabbit Reticulocyte Lysate 25.0 25.0
Amino Acid Mixture, Minus Leucine, 1mM 0.5 0.5
Amino Acid Mixture, Minus Methionine, 1mM 0.5 0.5
RNase Out (RNase inhibitor) 1.0 1.0
pGEM47-CREB DNA template ( add 1ug) 1.0 0.0
T7 RNA Polymerase 1.0 1.0

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 EMSA PROTOCOLS FIR 2010
7. Incubate the reactions for 90 minutes at 30°C.

8. Store at -20oC.

Note that while we do not have time to confirm the protein synthesis, typically you
would do this by visualizing by Western blot analysis or SDS-Page. See example
below.

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 EMSA PROTOCOLS FIR 2010

Preparation of Non-Denaturing Polyacrylamide (4%) Gel

1. Assemble the glass plates.

a. Make sure the glass plates, spacers, and combs are clean and free of dried gel
fragments, grease, and dust. Plates are washed with soap, rinsed with water followed by
a 70% ethanol solution, and dried with a kimwipe. Same for the spacers and combs.

b. Lay the large glass plate on a clean surface. Place one side spacer along each short
edge with the foam blocks at the top. Interlock the bottom spacer with the side spacers.

c. Place the small glass plate on top of the spacers so that both sides and the bottom of the
plates and the spacers are even. Press the foam blocks at the tops of the side spacers
firmly against the top edge of the short glass plate and clamp the assembly together with
two clips over each side spacer and two over the bottom spacer.

d. Confirm there are no gaps between the side spacer pads and the small plate and that the
bottom and side spacers are still interlocked. If there is a gap between the foam pads
and the top of the small plate or between the side and bottom spacers, buffer will leak out
of the upper buffer reservoir during electrophoresis.

2. Except for TEMED, mix reagents for a 4% non-denaturing polyacrylamide gel (see solutions
sheet).

3. Attach a 0.4 micron filter to a 60cc syringe with the plunger removed. Mount the syringe-
filter to the mouth of a 250ml side-armed flask using parafilm to keep it in place and to seal
the area around the filter.

4. Pour the acrylamide solution into the syringe and filter it by applying a vacuum to the side-
arm of the flask to pull the solution through the filter and into the flask.
5. After the solution has been filtered, remove the syringe-filter and place a rubber stopper on
the mouth of the flask and de-gas the solution for ~10 minutes by applying a vacuum.
6. Add TEMED, swirl the flask to thoroughly mix, and then carefully pour the acrylamide
solution between the assembled plates, taking care not to generate bubbles. Fill the
assembly to the top of the short plate. Dislodge any small bubbles by gently tapping the
glass and carefully insert the comb, making certain not to trap bubbles under the comb wells.
Allow the gel to polymerize completely before removing the comb (at least 1 hour).
7. Let any remaining solution polymerize in a 50 ml conical and use it to monitor the status of
polymerization. After polymerization is complete, dispose of the polymerized solution and
tube in the trash.

After polymerization, gels can be stored upright at 4oC overnight or several days by
covering the top (over the comb) with a wet paper towel and plastic wrap to prevent
dehydration (leave the comb, spacer and clamps in place).

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 EMSA PROTOCOLS FIR 2010

Prepare and Resolve Binding Reactions

1. 1X Tris-Glycine-EDTA (1X TGE) running-buffer has been prepared for you and is stored in
the 4oC cold room. This is also where you will run your gels.

2. Slowly lift the comb from the gel, being careful to maintain the structure of the wells and not
tear the gel dividing the lanes. Also remove the bottom spacer from the gel.

3. Place the gel into the electrophoresis apparatus, transfer it to the cold room, and fill
apparatus with the cold 1X TGE running buffer. Use a pipette to rinse the wells thoroughly
with running buffer and to dislodge any bubbles collected at the bottom of the gel.

4. Pre-run the gel at 100V at 4oC. During this time prepare your binding reactions.

5. Set up binding reactions.

a. Make your 2X EMSA binding buffer (recipe on the solutions’ sheet).

b. Label your tubes according to the table and place them on ice and keep them on ice
while assembling the reactions.

c. Adding everything except the probe, assemble binding reactions according to the
volumes and order of addition given in the EMSA table.

d. Vortex briefly to mix and briefly centrifuge (tap spin) the tube to collect the reaction mix
into the bottom of the tube.

e. Incubate the reaction on ice for 10 minutes.

f. Add 50 fmoles probe (1μl) and vortex to mix (tap spin, if needed)

g. Incubate the reactions for an additional 30 minutes on ice

6. Load and run your samples on the gel

a. Turn off current to the gel and load the gel.

b. Clean wells and remove any bubbles from the bottom of the gel (described above).

c. Using a gel-loading pipette tip (i.e. narrow or flat-edged tip), carefully layer each reaction
onto the bottom of the well, ordering them as indicated in the EMSA table.

Loading is best viewed if you are at eye-level with the wells. Also, because the gel is now at room
temperature, condensation will accumulate on the plates, requiring that you wipe the plates with a kimwipe
during the time you are loading.

8
 EMSA PROTOCOLS FIR 2010
7. Run at the gel at 240V for 1 ¾ hours.
Migration can be monitored by the TNT reactions, which are visible due to the presence
of hemoglobin.

8. Remove the plates from the apparatus and lay them on a clean, dry absorbent pad.
Remember, the probe is sensitive to fluorescent light, so it may be necessary to wrap the gel
in foil.

9. Image gels on the Odyssey Imager.

a. Put gel in imager align in proper corner (0,0 coordinates)

b. Open the Odyssey Imaging Program
c. Select File – New
d. Save file as – Fir2009\Date\Group
e. Select Scan, OK, OK
f. The Scanner Console should come up
1. Under Scan: set preset to DNAGel
2. Under Scan Parameters: don’t change
3. Under Channels: increase intensity to 7.0 on each channel
4. Under Scan Area: select gel size by adjusting the red square
g. Click Start Scan

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 EMSA PROTOCOLS FIR 2010

Solutions for EMSA

HEGD (hypotonic buffer) - Nuclear Extract Preparation
Final Conc Stock soln Vol (ul) for 10 ml Box
25 mM 1M HEPES pH 7.9 250 RT
1 mM 1M DTT 10 -20⁰C
1.5mM 0.5M EDTA pH 8.0 30 RT
10% 50 % glycerol 2000 RT
1X *50X protease inhibitor 200 -20⁰C
H2O 7510 RT
HEGDK (extraction buffer) - Nuclear Extract Preparation
Final Conc Stock soln Vol (ul) for 2ml Box
25 mM 1M HEPES pH 7.9 50 RT
1 mM 1M DTT 2 -20⁰C
1.5mM 0.5M EDTA pH 8.0 6 RT
10% 50% glycerol 400 RT
0.5M 1.0M KCl 1000 RT
1X *50X protease inhibitor 40 -20⁰C
H2O 502 RT
2X CREB EMSA Buffer
Final Conc Stock soln Vol for 1ml 2X Box
10mM Tris (7.4) 1M Tris (7.4) 20.0 RT
50mM NaCl 5M NaCl 20.0 RT
1mM MgCl2 1M MgCl2 2.0 RT
0.5mM EDTA 0.5M EDTA 2.0 RT
5% Glycerol 50% Glycerol 200.0 RT
0.5mM DTT 1M DTT 1.0 -20⁰C
125ug/ml BSA 20mg/ml BSA 12.5 4⁰C
50 ug/ml dIdC 0.5mg/ml 200.0 -20⁰C
H2O 542.5 RT

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 EMSA PROTOCOLS FIR 2010

4% Non-Denaturing Polyacrylamide Gel

Final Conc Stock soln Vol (ml) for 50ml Box
4% 40% acrylamide/bis- 5.00 4⁰C
1X 10X Tris-Glycine 5.00 RT
0.5mM 0.5M EDTA 0.05 RT
10% Ammonium
0.05% 0.25 4⁰C
Persulfate (APS)
0.10% TEMED 0.05 4⁰C
H2O 39.65 RT
100mM 8-Bromo-cAMP Stock (MW = 430.1 g/mole)
For 0.5 ml Final For 1 ml Final
In sterile H2O 21.5 mg 43.01 mg
*Roche Complete Protease Inhibitor (50X Stock)
For 0.2 ml Final For 1 ml Final
*In sterile H2O 1 complete mini tablet 1 complete or 5
* Protease inhibitors from the 50X stock must be added to solutions just prior to
their use (i.e. added fresh). The final solution concentration of inhibitors is 1X.
o
Extra 50X stock can be stored at -20 C for subsequent use.

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 EMSA PROTOCOLS FIR 2010

Date: EMSA TABLE

Probe: CRE (consensus)
Vol 10 ug Vol 50
Lane & Vol H 20 Vol 2x Competitor (fold Vol Comp. Vol Ab
Antibody Protein Protein fmole
Rxn# (ul)** Buffer (ul) excess) (ul) (ul)
(ul) Probe (ul)
1 10.0 --- 0.0 --- 0.0 MA-10 NE 1.0
2 10.0 CRE (100X) 0.5 --- 0.0 MA-10 NE 1.0
3 10.0 Star CRE2 (200X) 1.0 --- 0.0 MA-10 NE 1.0
4 10.0 Star mCRE2 (200X) 1.0 --- 0.0 MA-10 NE 1.0
5 10.0 --- 0.0 CREB (C-21) 1.0 MA-10 NE 1.0
6 10.0 --- 0.0 P-CREB (Ser133) 1.0 MA-10 NE 1.0
7 10.0 --- 0.0 c-JUN (N) 1.0 MA-10 NE 1.0
8 10.0 --- 0.0 P-JUN (Ser63) 1.0 MA-10 NE 1.0
9 10.0 --- 0.0 --- 0.0 CREB TNT 5.0 1.0
10 10.0 --- 0.0 CREB (C-21) 1.0 CREB TNT 5.0 1.0
11 10.0 --- 0.0 P-CREB (Ser133) 1.0 CREB TNT 5.0 1.0
12 10.0 --- 0.0 --- 0.0 No DNA TNT 5.0 1.0
**Total reaction volume = 20ul

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 EMSA PROTOCOLS FIR 2010
Extra Protocols
Preparation of End-Labeled Probes
Probes for EMSA will be generated end-labeling double-stranded oligodeoxynucleotide with 32P,
using the enzyme T4 polynucleotide kinase (PNK). We will be use T4 PNK from New England
Biolabs (Cat.# M0201S). All precautions for working with radioactivity must be followed. See
section in supplementary materials.

1. Set water baths or heat blocks to 37oC and 75oC.

2. Add the following components, in the order indicated, to a 0.5 ml microcentrifuge tube.
Incubate reaction at 37oC for 30-45 minutes.

End-Labeling Reaction
Reagent Volume (μL)
H2O 4.5
2.5 pmoles ds CRE oligo (5 uM stock) 0.5
10X PNK buffer 1.0
32
PATP (3000Ci/mmol,10mCi/ml) 3.0
*T4 PNK (10 units/ul; New England Biolabs Cat# M0201S) 1.0
Total Volume 10.0
*10X T4 PNK Buffer = 0.7 M Tris-HCL (pH 7.6), 100mM MgCl2, 50mM DTT

3. While reaction is incubating, make your spin column for separating the labeled
oligodeoxynucleotide from unincorporated or free 32PATP. This is done by size exclusion
chromatography in a spin column, whereby the sample is centrifuged through a Sephadex
G-50 resin equilibrated in 1X annealing buffer (10mM Tris pH 7.5, 0.1M NaCl, 1mM EDTA).

4. Preparation of the spin column is done as follows:

a. Place a disposable filter-column into a 15 ml conical tube.

b. Fill the column with ~2ml of a Sephadex G-50 slurry equilibrated in 1X annealing buffer.

c. Centrifuge in a tabletop centrifuge; speed 3K, 4 minutes (~1500 RCF). The approximate
position for this speed is marked on the centrifuges.

d. Discard the flow-through.

e. Add 100ul of 1X Annealing buffer to the top of the matrix in the column and centrifuge as
above.

f. Repeat “e” twice more.

g. Place the column into a new 15ml conical tube. It is now ready to use for nucleotide
separation.
Note: columns can be made before hand and stored at this point for several weeks at 4oC. However,
before use they will need to be re-equilibrated 13
with buffer (i.e. repeat “e” 1-2X more) before using.
 EMSA PROTOCOLS FIR 2010
5. After your probe reaction has incubated for the appropriate time, add 90ul of 1X annealing
buffer to the 10ul probe reaction. The EDTA in the annealing buffer will inactivate the
enzyme (alternatively, the enzyme can be heat inactivated by incubation at 75oC for 10
minutes).

6. Remove 1ul of the diluted probe and place into a vial (labeled sample 1) containing ~5ml
scintillation fluid

7. Using your size-exclusion column, purify your radiolabeled oligodeoxynucleotide from the
unincorporated nucleotides, as follows.

a. Carefully layer the probe sample onto the top of the G-50 matrix prepared in the column.
Be sure to layer it onto the center of the matrix so that it does not leak along the sides of
the column.

b. Centrifuge in a tabletop centrifuge exactly as you did for equilibrating the column above
(i.e. 3K, 4 minutes).
During centrifugation, the smaller nucleotides enter pores of the matrix, which slows their movement
through the column. Thus, the unincorporated 32PATP is retained in the column, while the larger
sized oligodeoxynucleotide, which is excluded from the matrix pores, moves rapidly through the
column and elutes into the 15 ml conical tube (see figure below). A green dye is added to the
nucleotides, and should remain near the top of the column. If it comes through with your eluate into
the conical tube holding the column, unincorporated nucleotides will be mixed with your probe.

c. Remove the column from the tube and discard

into the 32P waste container behind the
protective shield. The column will be very
radioactive, so take all precautions for using
radioactivity.

d. Working behind a Plexiglas shield, use a

micropipet to remove the probe from the bottom
of the 15ml conical and place it into a clean 1.5
ml microcentrifuge tube labeled with the group
name and date and for radioactive content.
Place it into the Plexiglas container.

e. Remove 1ul of the eluted probe into a second vial (labeled sample 2) containing ~5ml
scintillation fluid. Store remainder of the probe at -20oC.

f. Determine the amount of radioactivity initially added to the reaction (sample 1) and the
amount incorporated into your DNA (sample 2).
Calculate the percent incorporation [(sample2 ÷ sample1) X 100. This should be around 20%. Anything less
than 10% probably did not work. Less than 5% definitely did not work. Should this happen, ask the TA for a
probe previously prepared for the EMSA.

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 EMSA PROTOCOLS FIR 2010
Extra Protocols

Drying and Film Imaging of Radioactive EMSA Gels

1. Remove the plates from the apparatus and lay them on a clean, dry absorbent pad. Pry off
the top plate, by carefully twisting either one of the spacers or a spatula (or similar object)
inserted between the plates until the plates come apart. It is important that you are very
careful not to dislodge the gel from the bottom plate or cause it to wrinkle or pucker, as this
will distort the gel’s image when exposed to film.

2. Take two sheets of Whatman 3M or similar filter paper and place them on the gel. Peel the
paper with the gel attached away from the lower plate. Turn the gel over and place it, filter
side down, on the gel dryer.

3. Cover the gel with a piece of Saran wrap and dry for at least 1 hour at 80oC (2 hours at 60o-
70oC is better, if time permits).

4. When the gel is dry, take it, together

with a film cassette, a box of x-ray
film, and an intensifying screen, to the
darkroom. Place the dried gel,
together with an intensifying screen
and x-ray film, into a film cassette (see
figure for autoradiography set up).

5. Place cassette at -80oC for several hours to overnight.

6. Develop film.

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 CHIP PROTOCOLS FIR 2010
Experiment 2: Chromatin Immunoprecipitation to Evaluate Effects of Hormone Treatment
on Transcription Factor Binding In Vivo

Preparation of MA-10 Chromatin

1. Warm the cell culture media, Dulbecco’s Phosphate Buffered Saline (DPBS), and trypsin
solutions to 37oC in a water bath.

Emily and Valentine will prepare five 150mm culture dishes of MA-10 cells, using the
same treatments as for EMSA.
o Group A = no treatment control (vehicle only)
o Group B = + 1mM cAMP
o Group C = + 10-8M DHT
o Group D = + 10-8M DHT + 1mM cAMP
Results will be compared between groups.
All indicated volumes are for a single 150mm dish.

2. Isolate MA-10 cells from one 150mm culture dish as follows:

a. Remove media from cells by aspiration

b. Add 6 ml DPBS (w/o Ca+2 & Mg+2), swirl it over the cells to wash away any remaining
media, then remove by aspiration. Note, MA10 culture media has serum, which
strongly inhibits trypsin. Washing the cells is
c. Repeat “b” needed to remove the serum prior to trypsin

d. Add 8ml 0.05% trypsin. Incubate at room temperature for 45 seconds.

e. Remove trypsin solution by aspiration and incubate cells until you see them start to
dislodge from the plate when you tap it on the side with your palm. This will be between
1-2 minutes.

IMPORTANT: If you notice a large number of cells sloughing from the plate, do not
continue the incubation but proceed directly to the next step – f

f. Add 10ml complete media and, using a 10ml pipette, pipette the cells up and down,
rinsing the plate as you go, dislodging any remaining adherent cells.

g. Pipette cells into a 50ml conical tube and add an additional 20ml of complete media to
the plate, rinse the plate with the media, and pipette the media (and any remaining cells
on the plate) into the 50ml tube of cells.

h. Centrifuge for 5 min, 600xg, at room temperature to pellet cells.

i. Remove media and replace with 30 ml fresh media. Disperse cells by pipetting them
several times with a 10ml pipette.
 CHIP PROTOCOLS FIR 2010
3. Crosslink proteins and DNA in the MA-10 cells by adding 833uL 37% formaldehyde (final
formaldehyde concentration is 1%).

4. Invert to mix and incubate 10 min at 37oC.

5. Add 1.55ml 2.5M glycine (final conc. = 125mM) to stop, or quench, the cross-linking reaction.

6. Pellet cells by centrifugation (5 min at 600xg) at 4oC and aspirate media, removing as much
of the media as possible.

Note that until the crosslinks are reversed all steps must be performed at 4oC, as elevated
temperatures will induce crosslink reversal.

7. Wash the cells 2X with 10ml ice-cold 1X PBS containing 1X protease inhibitors (from 50X
stock).

8. Collect the cells by centrifugation (5 min at 600xg, 4oC).

9. Remove PBS by aspiration, making sure not to disrupt the cell pellet.

10. Re-suspend the cell pellet in 1.6ml ChIP lysis buffer and transfer 800ul to two chilled 1.5ml
microcentrifuge tubes (FYI- volumes should not exceed 1 ml per tube).

Prior determination of cell number is important to assure consistent chromatin preparations and
immunoprecipitation results. Normally, one would first calculate the number of cells in their sample.
The average number of MA-10 cells (at confluency) on one 150mm plate was previously determined
as ~4x107 and this number was used to determine the volume of ChIP lysis buffer, calculated as 200ul
ChIP lysis buffer per 5x106 cells.

Note, for ChIP to be successful, several parameters must be optimized. These were previously
determined for this assay and include cross-linking parameters, sonication conditions, PCR, and
immunoprecipitation with each antibody.

11. Incubate on ice for 10 minutes. Avoid longer incubations to prevent SDS from coming out
of solution.

12. Shear DNA by sonication.

a. Set the sonicator power to 40% (a power setting of greater than 50% may cause foaming
of the lysis buffer and reduce the shearing efficiency).

b. Place the sonication probe into the 800ul of lysate from above.
 CHIP PROTOCOLS FIR 2010
c. Keeping the sample on ice, pulse for 10 seconds and then rest (on ice) for at least 2
minutes. Repeat with the next sample.

IMPORTANT: Sonication causes rapid heating, which can reverse formaldehyde crosslinks and
damage your sample. To limit sample heating, perform this step on ice using intermittent
pulses, followed by incubation on ice for 2-4 minutes (or dry ice for shorter times).

Sonication conditions, for sheering DNA to a size range of 200bp-1000bp (avg. size 500bp),
were previously determined by varying the power and pulse number.

d. Repeat “c” 19 more times, to make a total of twenty, 10 second pulses, each with an
intermittent incubation on ice.

13. Clear any cellular debris from the sonicated lysates by centrifugation in a microcentrifuge.
(14,000 rpm, or about 16,000xg, 10 min, 4oC).

14. Transfer supernatant to a chilled microcentrifuge tube or, if prior to sonication the samples
were divided into aliquots, combine similar samples into a chilled 15ml conical tube and mix
well by pipetting up and down.

To reduce the time needed to process everyone’s samples, groups should coordinate sonicator use. It
usually works well if groups work concurrently; taking turns to sonicate samples. Thus, one group
sonicates and while their samples rest on ice another group sonicates their samples. By the time all
groups have sonicated, it is time again for the first to perform their next sonication.

15. Remove a 10ul aliquot of undiluted chromatin and place it in a new 1.5ml microcentrifuge
tube. Label this tube “input control” and store at -80oC for use later.

16. Measure the total volume of remaining chromatin and add a 9x volume of ice-cold ChIP
Dilution Buffer (i.e. for each 100ul of the sheared chromatin add 900ul ChIP Dilution Buffer).
Using the entire batch of diluted chromatin, aliquot 1ml samples into 1.5ml microcentrifuge
tubes.

One, 1ml aliquot will be used for each immunoprecipitation. FYI, the chromatin can be used
immediately or stored at –80oC for several weeks.
 CHIP PROTOCOLS FIR 2010

Immunoprecipitation
1. Pre-clear chromatin (if the diluted chromatin is frozen, thaw it on ice)

In subsequent steps, antibody-antigen complexes will be precipitated with Protein A-conjugated agarose
beads. The beads can bind free genomic DNA that is not part of the precipitated immune complex and, if
not controlled for properly, will contribute substantially to eluted fractions containing the desired specific
protein-DNA complexes. To reduce the amount of contaminating nonspecific DNA, diluted chromatin
samples are first pre-cleared of free genomic DNA by incubation with agarose beads.

a. You will need one 1ml aliquot of diluted chromatin for each immunoprecipitation to be
performed. Using wide-orifice pipette tip, add 75ul of a 50% slurry of pre-blocked protein
A-agarose beads (purchased from Upstate, 16-157, in 4°C box) to each chromatin
sample. Mix beads well before pipetting.

b. Incubate 90 min at 4oC on rotator.

c. Pellet the agarose beads by microcentrifugation at 6500 RPM (~3800xg) for 1 min at 4oC.

d. Remove chromatin-containing supernatant to a new pre-chilled 1.5ml microcentrifuge

tube.
ChIP Sample (Antibody) Table
Vol. Ab
Rxn # Student Antibody AB source μL Ab type (concentration) Ab Conc (mg/mL)
1 1 CREB (C‐21) Santa Cruz sc‐186 25 ul IgG rabbit polyclonal (200 ug/ml)
2 IgG Control Santa Cruz sc‐2027 12.5 ul IgG rabbit normal (0.4 mg/ml)
3 2 P‐CREB (Ser133) Upstate 06‐519 5 ul IgG Rabbit polyclonal (1 mg/ml)
4 CBP Upstate (Millipore) 06‐297 1.47 ul IgG rabbit Polyclonal (200 μg; 3.39 mg/ml)
5 3 c‐JUN (N) Santa Crux sc‐45 25 ul IgG rabbit polyclonal (200 ug/ml)
6 SF‐1 ABR PA1‐800 5 ul IgG rabbit (1 mg/ml)
7 4 P‐JUN (Ser63) Upstate (Millipore) 06‐828 10 ul IgG rabbit polyclonal (100μL)
8 Pol II Control Covance MMS‐126R 2.5 ul IgG mouse monoclonal (2‐3 mg/ml)

2. Each group will perform the immunoprecipitations listed in the ChIP Sample Table above,
with each student responsible for a reaction set.

3. Add the volume indicated (ChIP Sample Table) for 5ug primary antibody to each pre-cleared
1ml sample of diluted chromatin. See TA’s for antibodies.

4. Rotate the samples at 4°C overnight.

5. Add 60ul of protein A-agarose slurry (Upstate, in 4°C Box) to each tube to sequester the
immune complexes. Mix bead well before pipetting and use a wide-orifice pipette tip.

6. Rotate for 1 hour at 4°C. During the incubation, prepare wash solutions and chill them on
ice.
 CHIP PROTOCOLS FIR 2010
7. Collect the protein A-agarose beads (contains immune-complexes) by microcentrifugation for
2 min at 3000 RPM (~300xg), rt.

8. Making sure not to disturb the pellet, use a 1ml pipetman to remove as much of the
supernatant as possible. Discard supernatant.

9. Add the first wash buffer (see Step 10) to the beads, mix by pipetting, and transfer to a
centrifuge tube filter unit.

10. Perform the following washes with rotation; 5 min each at rt. Do not vortex the beads to mix.
Instead, tap or invert them. Between each wash, pellet the agarose beads, as above, and
discard the flow-through.

a. 1X - 500µl ChIP Low-Salt Wash Buffer

b. 1X - 500µl ChIP High-Salt Wash Buffer
c. 1X - 500µl ChIP LiCl Wash Buffer
d. 2X - 500µl ChIP TE

11. Centrifuge again and remove any excess liquid.

12. Make Elution Buffer (100mM NaHCO3 + 1% SDS). This must be fresh.

13. Elute immunoprecipitated complexes

a. Place the columns in a new, dolphin-nosed tube.

b. Add 100ul of elution buffer to each sample of beads
c. Flick or tap the tube to resuspend the beads
d. Incubate samples at room temperature for 15 min with rotation.
e. Centrifuge for 1 min @ 6,500 RPM (~3800RCF).
f. Repeat elution (200ul total elution).
g. Discard the column.
h. Add 8ul 5M NaCl (200mM final concentration) to the pooled sample. Vortex to mix.
i. Add 190ul of elution buffer to the 10ul chromatin aliquot previously saved for the
input control (from step 12 of “Prepare the MA10 Chromatin” above). Mix and add
8ul 5M NaCl. Vortex.
 CHIP PROTOCOLS FIR 2010

Reverse Cross-links and purify DNA

1. Reverse formaldehyde cross-links of all samples and the input control (stored at -80oC) by
incubating them at 65oC overnight. [Note, the 65oC incubation can be shortened but should
extend at least 4hr]

2. Add 4ul 0.5M EDTA, 10ul 1M Tris pH 6.5, 1.6ul Proteinase-K (10 mg/ml PK in H2O, in -20°C
box) to each sample (final concentration equals 80ug/ml PK, 50mM Tris, 10mM EDTA).

3. Incubate at 45oC for 1-1½ hr.

4. Extract samples using the ChIP DNA Clean & Concentrator Kit from Zymo Research. (see
notes at the end of this protocol for steps on Phenol-chloroform extraction)

a. Add 1.118ml ChIP DNA Binding Buffer to each volume of sample (5:1). Mix briefly.
b. Transfer mixture to a provided Zymo-Spin™ Column in a Collection Tube.
c. Centrifuge at ≥10,000 x g for 30 seconds. Discard the flow-through.
d. Add 200ul Wash Buffer to the column. Centrifuge at ≥10,000 x g for 30 seconds.
e. Repeat wash step.
f. Add 50ul Elution Buffer directly to the column matrix. Transfer the column to a new
1.5ml tube and centrifuge at ≥10,000 x g for 30 seconds to elute DNA.
g. DNA is now clean and ready to use for PCR.

This, and similar column-based DNA purification, use a column containing a silica based
membrane or matrix that is position in a spin column or tube that is designed for convenience
to fits into a microcentrifuge. High ionic strength buffer (binding buffer) is added to the DNA.
The DNA is then placed into the spin column and centrifuged, forcing it into the silica-based
membrane. The chaotropic salt added to the DNA drives its binding to the silica by forming a
salt bridge between the negative charges on DNA (phosphate groups) and silica (oxygen
anion). Thus, under conditions of high ionic strength, DNA binds to the silica on the
membrane. The binding is stable and the DNA sample can be cleaned by washing the
column with buffer that maintains the salt bridge. The clean DNA is then eluted from the
column using a low salt buffer, which disrupts the salt bridge.
 CHIP PROTOCOLS FIR 2010

qPCR Analysis of ChIP DNA

1. Thaw primers at room temperature.

2. Using filtered pipette tips (to reduce the chance of contamination), assemble two PCR
cocktails: one for the StAR proximal promoter (rxns 1-11) and the other for the StAR distal
region (rxns #12-22), which is located 4.5kb upstream of the promoter and serves as a
control for DNA shearing. Each reaction will be run in triplicate, so for each set of 11
reactions, make a 36X cocktail. This assures sufficient reaction mixture for all samples,
should there be any pipetting errors. To make the cocktail, add the reagents in the PCR
Reaction Mix Table, in the order given, to a 1.5μl microcentrifuge tube and mix.

PCR REACTION MIX TABLE

Reagent Conc/rxn Vol/25µl rxn (µl) Vol/36 rxns (µl) Box
Water 9.5 342.0 RT
2x Sybr Green 1x 12.5 450.0 4°C
For Primer 250nM 1.0 36.0 -20°C
Rev Primer 250nM 1.0 36.0 -20°C
DNA template 1.0 -20°C
Prox For: STAR proximal forward: 5'-TGATGCACCTCAGTTACTGG-3' Tm=53.8
Prox Rev: STAR proximal reverse: 5'-GCTGTGCATCATCACTTGAG-3' Tm=54.1
Distal For: STAR distal forward: 5'-CATACGTGCACTGTCTTAGC-3' Tm=51.4
Distal Rev: STAR distal reverse: 5'-ACTCCTCCAGTAACTCCTTC-3' Tm=49.7
*Make two cocktails: one containing the For and Rev primers for the proximal promoter and
other with the For and Rev primers for the distal promoter

3. Aliquot 24µl of the StAR promoter PCR cocktail into rows A-D on your 96-well plate, as
shown in the PCR Setup Table below. Aliquot 24µl of the negative control (distal promoter)
cocktail into rows E-H.
 CHIP PROTOCOLS FIR 2010

4. Add the template DNA to each well, as shown in the table.

PCR PLATE SETUP

1 2 3 4 5 6 7 8 9 10 11 12
CREB (C-21) IgG control p-CREB (Ser133)
A Prox. promoter Prox. Promoter Prox. Promoter

CBP c-Jun (N) SF-1

B Prox. promoter Prox. Promoter Prox. promoter

p-Jun (Ser63) Pol II control Genomic DNA

C Prox. promoter Prox. promoter Prox. Promoter

Input control Water

D Prox. promoter Prox. Promoter

CREB (C-21) IgG control p-CREB (Ser133)

E Distal region Distal region Distal region

CBP c-Jun (N) SF-1

F Distal region Distal region Distal region

p-Jun (Ser63) Pol II control Genomic DNA

G Distal region Distal region Distal region

Input control Water

Distal region Distal region
H

5. Seal the plate tightly, centrifuge on a tabletop centrifuge for 2 min at 1000 x g, and place in
the Real-Time PCR machine (StepOne Plus, Applied Biosystems).

6. PCR amplify the DNA samples using the cycle parameters shown below. The program is
already set in the PCR machine but the denaturing step may need to be added.

PCR CYCLING CONDITIONS

Stage Temperature & Time Cycles
One – UNG Activation 50°C for 2 minutes 1
Two – Initial Denaturation 95°C for 10 minutes 1
Three
Denaturation 95°C for 15 seconds 40
Annealing 60°C for 15 seconds
Four – Dissociation 95°C for 15 seconds 1
60°C for 15 seconds 1
95°C for 15 seconds 1
Expected Product Size for Proximal Promoter (Prox): 145bp
Expected Product Size for Distal Promoter (Dist):156bp
 CHIP PROTOCOLS FIR 2010

Solutions for ChIP

FINAL CONC STOCK CONC
ChIP Lysis Buffer Vol (ul) for 2ml Final Storage Box
10mM 0.5M EDTA 40 RT
50mM 1M Tris pH 8.1 100 RT
1% 10% SDS 200 RT
1X *50X complete protease inhibitor 40 -20
H2O 1620
ChIP Dilution Buffer Vol (ul) for 15ml Final Storage Box
16.7mM 1M Tris pH 8.1 251 RT
167mM 5M NaCl 502 RT
1.22mM 0.5M EDTA 37 RT
1.10% 10% Triton X-100 1.65 ml RT
0.01% 10% SDS 15 RT
1X *50X complete protease inhibitor 300 -20
H2O 12.25 ml
ChIP Low-Salt Wash Vol (ul) for 5ml Final Storage Box
20mM 1M Tris pH 8.1 100 RT
150mM 5M NaCl 150 RT
2mM 0.5M EDTA 20 RT
1% 10%Triton X-100 500 RT
0.10% 10% SDS 50 RT
1X *50X complete protease inhibitor 100 -20
H2O 4080
ChIP High-Salt Wash Vol (ul) for 5ml Final Storage Box
20mM 1M Tris pH 8.1 100 RT
500mM 5M NaCl 500 RT
2mM 0.5M EDTA 20 RT
1% 10% Triton X-100 500 RT
0.10% 10% SDS 50 RT
1X *50X complete protease inhibitor 100 -20
H2O 3730
ChIP LiCl Wash Buffer Vol (ul) for 5ml Final Storage Box
250mM 5M LiCl 250 RT
1% 10% NP-40 500 RT
1% 10% Na Deoxycholate 500 RT
10mM 1M Tris pH 8.1 50 RT
1mM 0.5M EDTA 10 RT
1X *50X complete protease inhibitor 100 -20
H2O 3590
ChIP TE Vol (ul) for 10ml Final Storage Box
10mM 1M Tris pH 8.0 100 RT
1mM EDTA 0.5M EDTA 20 RT
1X *50X complete protease inhibitor 200 -20
H2O 9680
ChIP Elution Buffer Vol (ul) for 2ml Final Storage Box
100mM 1M NaHCO3 200 RT
1% 10% SDS 200 RT
H2O 1600
Roche Complete Protease Inhibitor (50x stock) For 1 ml Final
Make up in Sterile Water 1 complete or 5 complete mini
*Protease inhibitors from the 50x stock must be added to the solutions just prior to their use. Extra 50x stock can be
stored at -20°C for subsequent use.
 CHIP PROTOCOLS FIR 2010

Additional Protocols:
1. Phenol-chloroform extraction of ChIP DNA

This denatures and removes protein contaminants. An additional chloroform-only extraction

is performed to remove any excess phenol, which can inhibit subsequent enzymatic steps.

a. Add phenol:chloroform (Tris-saturated) to your sample at a ratio of 1:1 (v/v).

b. Cap the samples tightly and vortex thoroughly to mix (~15sec).
c. Microcentrifuge the samples for 5 min. at room temperature (14K RPM,~16Kxg)
d. Remove the upper aqueous phase to a new 1.5ml microcentrifuge tube. Be careful
not to contaminate the sample with components from the interface or the lower
organic phase.
e. Add chloroform to your sample at a ratio of 1:1 (v/v).
f. Cap tubes tightly and vortex ~15 sec.
g. Microcentrifuge samples, as in “c”.
h. Remove the upper aqueous phase to a new 1.5ml Microcentrifuge tube, taking care
not to carry over any of the lower organic phase.

2. Precipitation of DNA from the extracted aqueous phase

a. Add 2ul glycogen (10ug total) and 12ul 5M NaCl to the sample

Glycogen acts as a carrier to help bring down the DNA and salt is needed to neutralize
the charges on the DNA backbone. When ethanol is added in the next step, it
facilitates the formation of the ion pairs between the polyanion (DNA) and the cations
from the salt, causing it to precipitated from solution

b. Mix and then add 1ml 100% EtOH (-20oC pre-cooled) and mix again.
c. Place the samples at -80oC for 30 minutes (placing samples in the cold helps
facilitate precipitation but carries down more salt).
d. Centrifuge the samples for 30 minutes at 14K RPM (~16Kg) in a microcentrifuge at
4oC.
e. The DNA will be in a small pellet at the bottom of the tube. Being careful not to
dislodge the pellet, remove the supernatant with a pipette.
f. Add 250ul 70% EtOH to the pellet, mix gently, and centrifuge as above for 5 min.
(The 70% EtOH wash helps remove residual salts from the precipitated DNA).
g. Remove the 70% EtOH wash solution, again taking care not to dislodge the pellet.
Try to remove as much of the ethanol solution as possible.
h. Air dry the DNA for ~15 minutes to evaporate any remaining ethanol

The pellets from ChIP DNA are often very loose and easily dislodged from the tube wall.
Leaving a small amount of EtOH is advisable rather that risking loss of the samples. Longer
air drying times or vacuum desiccation can be used to remove excess ethanol.

i. Except for the input control, re-suspend each pellet in 50ul of nuclease-free H2O.
Re-suspend input control in 250ul H2O.
j. Incubate samples at 55oC for 5-10 minutes to facilitate DNA solubilization.