Moss Tom. DNA-protein interactions: principles and protocols
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Combined SW–Nuclease Footprinting 139
5. Ammonium persulfate (10%; w/v): Dissolve (by vigorous vortexing) 1 g of
ammonium persulfate (Bio-Rad) in 10 mL of distilled, deionized water. Filter
through a 0.22-µm membrane filter. Store at 4°C; make fresh weekly. Ammo-
nium persulfate is extremely destructive to tissue of the mucous membranes and
upper respiratory tract, eyes, and skin. Inhalation may be fatal. Exposure can
cause gastrointestinal disturbances and dermatitis. Wear gloves, safety glasses,
respirator, and other protective clothing and work in a chemical fume hood. Wash
thoroughly after handling.
6. SDS–polyacrylamide gel electrophoresis (PAGE) running buffer (10X stock):
0.25 M Tris base (Sigma) and 1.92 M glycine (Sigma). It is not necessary to
adjust the pH. The proportions of Tris base and glycine give pH 8.3. Store at
room temperature in a large vessel (carboy). Dilute to 1X with distilled, deion-
ized water, then add SDS to a final concentration of 0.1% (w/v).
7. SDS-PAGE sample buffer (4X stock): 0.5 M Tris–HCl pH 6.8, 8% (w/v) SDS,
40% (v/v) glycerol (Sigma), and 0.4% (w/v) bromophenol blue (Sigma). Store in
aliquots at –20°C. Before use, make a working solution of SDS-PAGE sample
buffer by diluting the 4X stock buffer and adding 2-mercaptoethanol (Sigma)
to a final concentration of 5% (v/v) (see Note 1). 2-Mercaptoethanol is harm-
ful if inhaled or absorbed through the skin. High concentrations are extremely
destructive to the mucous membranes, upper respiratory tract, skin, and eyes.
Use only in a chemical fume hood. Gloves, safety glasses, and respirator should
be worn.
8. Western transfer buffer: 50 mM Tris base, 380 mM glycine, 0.1% (w/v) SDS, and
20% (v/v) methanol (see Note 2). There is no need to adjust the pH of this buffer
by the addition of acid or alkali. Store at room temperature in a large vessel.
9. Dithiothreitol (stock): Prepare a stock solution of 0.5 M dithiothreitol (Sigma)
in distilled, deionized water and store in aliquots at –20°C. (See safety note in
item 7.)
10. Phenylmethylsulfonyl fluoride (PMSF; stock): Prepare a 100 mM stock solution
of PMSF (Boehringer, Indianapolis, IN) in absolute ethanol and store in aliquots
at –20°C. PMSF is extremely destructive to the mucous membranes of the respi-
ratory tract, the eyes, and the skin. It may be fatal if inhaled, swallowed, or
absorbed through the skin. It is a highly toxic cholinesterase inhibitor. Therefore,
it should be used in a chemical fume hood and gloves and safety glasses should
be worn during handling.
11. 1 M KCl.
12. 0.5 M MgCl
2
.
13. SW blocking/renaturation buffer: 3% (w/v) nonfat dried milk (or 5% [w/v] lipid-
free bovine serum albumin [BSA]; see Note 3a), 25 mM HEPES·KOH, pH 7.5,
50 mM KCl, 6.25 mM MgCl
2
, 1 mM dithiothreitol (freshly added from the 0.5 M
stock solution immediately before use), 10% (v/v) glycerol, 0.1% (v/v) Nonidet
P-40 (BDH, London, UK), and 0.2 mM PMSF (freshly added from the 100 mM
stock solution just prior to use). Store at 4°C. Prepare also some SW blocking/
renaturation buffer minus dried milk (or BSA).
140 Papavassiliou
14. Poly(vinyl alcohol) (stock): Prepare a 10% (w/v) stock solution of poly(vinyl
alcohol) (Sigma P 8136; average mol. wt. = 10,000) in distilled, deionized water
and store at –20°C.
15. SW binding/washing buffer: 12.5 mM HEPES
.
KOH, pH 7.5, 50 mM KCl, 6.25 mM
MgCl
2
, 0.5 mM dithiothreitol (freshly added from the 0.5 M stock solution im-
mediately before use), 2% (w/v) polyvinyl alcohol, 10% (v/v) glycerol, 0.05%
(v/v) Nonidet P-40, and 0.2 mM PMSF (freshly added from the 100 mM stock
solution just prior to use). Store at 4°C. (See Note 4b.)
16. Phenol/chloroform/isoamyl alcohol (25:24:1; v/v): Mix just prior to use 25 vol
of phenol with 24 vol of chloroform and 1 vol of isoamyl alcohol. Phenol is highly
corrosive and can cause severe burns. Any areas of skin that come in contact with
phenol should be rinsed with a large volume of water or PEG 400 (Sigma) and
washed with soap and water (do not use ethanol!). Chloroform is irritating to the
skin, eyes, mucous membranes, and respiratory tract. It is also a carcinogen and
may damage the liver and kidneys. Wear gloves, protective clothing, safety
glasses, and respirator when handling these substances and carry out all manipu-
lations in a chemical fume hood.
17. Chloroform/isoamyl alcohol (24:1; v/v): Mix 24 vol of chloroform with 1 vol
of isoamyl alcohol. This organic mixture can be stored at room temperature in
dark (brown) bottles indefinitely.
18. Nonspecific competitor DNA (stock solution): Dissolve salmon/herring sperm or
calf thymus DNA in distilled water, deproteinize it by sequential phenol/chloro-
form/isoamyl alcohol (25:24:1; v/v) and chloroform/isoamylalcohol (24:1; v/v)
extractions, sonicate to reduce the mean DNA length to 100–200 bp, precipitate
the DNA with ethanol, then resuspend it at 1 mg/mL in distilled, deionized water.
2.1.2. Reagents/Special Equipment
1. N,N,N',N'-tetramethylethylenediamine (TEMED; Bio-Rad). TEMED is
extremely destructive to tissue of the mucous membranes and upper respiratory
tract, eyes, and skin. Inhalation may be fatal. Prolonged contact can cause severe
irritation or burns. Wear gloves, safety glasses, respirator, and other protective
clothing and work in a chemical fume hood (TEMED is flammable!). Wash thor-
oughly after handling.
2. Protein extract of interest (e.g., whole-cell-free extract, crude nuclear extract, or
partially purified extract), as concentrated as possible.
3. Prestained nonradioactive MW standards (Bio-Rad) or [
14
C]-methylated protein
MW markers (Amersham).
4. Nonfat dried milk powder (e.g., Cadbury’s Marvel or Carnation), or fatty acid-
free BSA (Boehringer; fraction V).
5. Phenol: Redistilled (under nitrogen) phenol equilibrated with 100 mM Tris-HCl,
pH 8.0, and 1 mM EDTA in the presence of 0.1% (w/v) 8-hydroxyquinoline.
Phenol can be stored at 4°C in dark (brown) bottles for up to 2 mo. See the rel-
evant safety note in Subheading 2.1.1.
6. Absolute ethanol.
Combined SW–Nuclease Footprinting 141
7. Salmon/herring sperm or calf thymus DNA (Sigma or Boehringer).
8. Singly
32
P end-labeled DNA probe bearing the binding site(s) of interest (see
Note 5). All necessary precautions should be observed to minimize exposure to
ionizing radiation during labeling and isolation of the probe; work behind protec-
tive screens whenever possible.
9. Radioactive ink: Mix a small amount of
32
P with waterproof black drawing ink,
to a concentration of approx 200 cps (on a Geiger counter) per microliter.
10. 0.22-µm membrane filters (Millipore, Bedford, MA).
11. Mini-slab gel electrophoresis apparatus, giving 0.5- to 1.0-mm-thick mini-gels
(e.g., Bio-Rad Mini Protean II system), and accompanying equipment.
12. High-current (2–3 A) power supply (e.g., Bio-Rad or Hoefer, San Francisco, CA)
and electroblotting apparatus for Western transfer (e.g., Bio-Rad Trans-Blot);
additional equipment for Western transfer (11).
13. Nitrocellulose membrane: suitable membranes comprised of unsupported or sup-
ported nitrocellulose are available from a number of manufacturers, such as
Schleicher & Schuell (Keene, NH; BA85, 0.45 µm), Millipore (Immobilon-NC),
and Amersham (Hybond-C/C extra).
14. Plastic trays.
15. Forceps.
16. Plastic wrap such as cling film or Saran Wrap
®
.
17. X-ray film (e.g., Kodak X-Omat AR, Rochester, NY).
18. Additional equipment: protective gloves/glasses/respirator, 4°C shaking air
incubator, sonicator, 25°C shaking air incubator, Geiger counter, Kimwipes, all
equipment for autoradiography.
2.2. Exposure of SW Blots to DNase I Treatment
2.2.1. Solutions
1. Eppendorf tube siliconization solution: 2% (v/v) dimethyldichlorosilane in 1,1,1-
trichloroethane (BDH). Eppendorf tubes should be silanized by briefly immers-
ing the opened tubes in a beaker containing this solution, pouring off excess
solution, and allowing the tubes to dry in air at room temperature. Dimethyl-
dichlorosilane is particularly toxic. Gloves, safety glasses, respirator, and other
protective clothing should be worn when handling it and should only be used in a
chemical fume hood.
2. Solutions 1, 12, and 15–17 of Subheading 2.1.1.
3. DNase I (stock solution): Dissolve DNase I in 50% glycerol (in distilled, deion-
ized water) to a concentration of 2.5 mg/mL. Store frozen in 10-µL aliquots at –20°C
or –70°C. This stock is stable indefinitely.
4. 1 M CaCl
2
.
5. DNase I reaction buffer: 10 mM MgCl
2
and 5 mM CaCl
2
. Store at room temperature.
6. DNase STOP solution A: 20 mM HEPES·KOH, pH 7.5, 20 mM EDTA, and 0.5%
(w/v) SDS. Store at 4°C.
7. 5 M NaCl.
8. DNase-STOP solution B: 60 mM HEPES
.
KOH, pH 7.5, 0.6 M NaCl, 60 mM
EDTA, 1.5% (w/v) SDS. Store at 4°C.
142 Papavassiliou
9. Proteinase K (stock solution): Dissolve Proteinase K in TE (10 mM Tris-HCl,
pH 7.4, and 1 mM EDTA) to a concentration of 2.5 mg/mL. Store in aliquots at –20°C.
10. Probe elution buffer: 20 mM HEPES·KOH, pH 7.5, 0.3 M NaCl, 3 mM EDTA,
0.2% (w/v) SDS, and 50 µg/mL Proteinase K.
11. Glycogen (stock solution): Prepare a stock solution of 10 mg/mL glycogen
(Sigma G 0885) in distilled, deionized water and store in aliquots at –20°C; gly-
cogen is used as a carrier to promote the precipitation of nucleic acids.
12. Ice-cold 80% (v/v) ethanol.
13. Formamide loading buffer: 90% (v/v) deionized formamide, 1X TBE (see below),
0.025% (w/v) xylene cyanol FF (Sigma), and 0.025% (w/v) bromophenol blue.
Store at –20°C after filtering. Formamide is a teratogen; take all safety precau-
tions to avoid contact during manipulations involving this reagent.
14. TBE buffer (5X stock): 445 mM Tris base, 445 mM borate, 12.5 mM EDTA.
Dissolve (under stirring for at least 1 h) 272.5 g ultrapure Tris base, 139.1 g boric
acid (Sigma), and 23.3 g EDTA·(Na
2
) dihydrate (Sigma) in 4.5 L of distilled,
deionized water. Make up to a final volume of 5 L. It is not necessary to adjust
the pH of the resulting solution, which should be around 8.3. Store at room tem-
perature in a large vessel. This stock solution is stable for many months, but it is
susceptible to the formation of a precipitate and should be inspected visually
from time to time.
15. Fixing solution: 10% (v/v) acetic acid and 10% (v/v) methanol.
2.2.2. Reagents/Special Equipment
1. DNase I (DPFF grade; Worthington, Freehold, NJ).
2. Proteinase K (Boehringer).
3. Absolute ethanol (at –20°C).
4. Single-edged disposable razor blades.
5. Forceps.
6. Siliconized 0.5-mL Eppendorf microcentrifuge tubes.
7. Siliconized 1.5-mL Eppendorf microcentrifuge tubes.
8. Drawn-out Pasteur pipets.
9. Whatman (Clifton, NJ) 3MM paper.
10. Items 16 and 17 in Subheading 2.1.2.
11. Intensifying screen (e.g., Cronex Lightning Plus; DuPont, Wilmington, DE).
12. Additional equipment: beaker, sharp-tip pencil, wet ice, scintillation vials/
counter, vortexer, electronic timer, microcentrifuge (Eppendorf or equivalent),
vortexing shaker set at 37°C, Geiger counter, SpeedVac concentrator (Savant,
Hicksville, NY), thermostatted heating block at 95°C, plastic tank at gel dimen-
sions (for gel fixing), vacuum gel dryer, and all equipment for autoradiography.
3. Methods
3.1. SW Blotting
The SW protocol involves four steps: electrophoretic separation of proteins
by SDS-PAGE, electroblotting of the gel-fractionated proteins onto nitrocellu-
Combined SW–Nuclease Footprinting 143
lose (NC) membrane, probing of the blocked blot with the desired DNA probe,
and detection of bound DNA by autoradiography of the washed, wet membrane.
1. Prepare and load the sample(s) and protein MW markers onto a standard SDS–
polyacrylamide gel (11,12) and electrophorese at an appropriate voltage until the
bromophenol blue dye reaches the bottom of the gel. An 8–10% polyacrylamide
separating gel is capable to resolve throughout most of the known DNA-binding
transcription factor size range. Because the strength of the final signal obtained
by the SW technique is proportional to the quantity of protein electrophoresed on
the gel, best results are obtained by running the maximum amount of extract that
does not overload the gel. For typical 0.5- to 1-mm-thick protein mini-gels, this
is usually 30–150 µg of whole-cell-free, crude nuclear, or partially purified extract
protein per lane. (See Note 1.)
2. Remove the electrophoresed gel from the glass plates. Assemble a Western blot
sandwich, place it in the electrophoresis tank containing an appropriate volume
of Western transfer buffer, and electroblot the proteins in the electrophoresed gel
onto a NC membrane according to standard Western blotting protocols (11,13).
Bear in mind that the best protein transfer is usually achieved by longer transfer
times (i.e., 30 V [40 mA] overnight at 4°C). (See Note 2).
3. When transfer is complete, gently peel the NC membrane off the gel, place it in a
plastic tray, and gently wash for 10 min with 20–30 mL of SW blocking/renatur-
ation buffer, omitting dried milk (or BSA).
4. Decant the solution and replace it with a sufficiently large volume of SW block-
ing/renaturation buffer to completely immerse the membrane (usually 30–50 mL).
5. Incubate overnight at 4°C with gentle rocking or shaking to block nonspecific
binding sites on the membrane and to allow renaturation of the filter-immobi-
lized proteins. (See Note 3.)
6. Using forceps, transfer the membrane to a fresh plastic tray and gently wash
for 5 min with 20–30 mL of SW binding/washing buffer. The membrane can be
stored in this buffer at 4°C for up to 1 d before incubation with the DNA probe.
7. Immerse the membrane (using forceps) in a fresh plastic tray containing a radio-
active probe mixture consisting of the following:
• SW binding/washing buffer (see Note 4).
• 5–10 µg (specific activity approx 10
7
cpm/µg) of an asymmetrically [
32
P]-
labeled DNA fragment bearing the recognition site(s) for the sequence-spe-
cific DNA-binding factor(s) of interest (see Note 5).
•20µg/mL nonspecific competitor DNA (see Note 6).
To make the probe concentration as high as possible, the volume of SW bind-
ing/washing buffer should be the minimum needed to cover the membrane fully
(smallest volumes are achieved if the membrane is sealed in a plastic bag). Work
behind Perspex or glass shields!
8. Incubate for 2–4 h at room temperature with gentle rocking or shaking. Longer
incubation times at a lower temperature may result in a better signal from a poorly
binding factor.
144 Papavassiliou
9. Carefully remove the radioactive probe mixture and dispose of it safely (work
behind Perspex or glass shields!).
10. Using forceps, transfer the membrane to a plastic tray and wash for 10 min with
100 mL of SW binding/washing buffer on a platform shaker (room temperature).
11. Repeat step 10 two to three times or until the radioactive level of the membrane
no longer falls appreciably between washes (this can be monitored by a Geiger
counter). (See Note 7.)
12. Place the wet NC sheet between two layers of tightly drawn cling film. With a
pad of Kimwipes, push out any trapped air bubbles under the cling film.
13. Expose to X-ray film at 4°C to locate regions of radioactive signal (protein[s]-
bound probe). Exposure times of 1–3 h are usually sufficient to detect the radio-
active species on the membrane. (See Note 8.)
3.2. Exposure of SW Blots to DNase I Treatment
The in situ footprinting reaction is done in four stages: localization and
excision of the areas on the NC sheet corresponding to protein-bound probe,
partial digestion of the individual strip-associated and control (free in solution)
DNAs with DNase I, extraction of the protein-bound DNA from the strip-
immobilized protein–DNA complex, and analysis of the free and complexed
DNA digestion products on a DNA sequencing gel.
1. Following autoradiography, align the NC sheet with the autoradiogram (see
Note 8), mark the precise position of radioactive signal(s) with a sharp-tip pencil
(this will permit determination of the relative molecular weight of the detected
DNA-binding factor[s]), and cut the strip(s) corresponding to radioactive
signal(s) with a clean, sharp razor blade.
2. Using forceps, uncover the strip from the cling film and immediately transfer it
into a siliconized 0.5-mL Eppendorf tube containing 200 µL of SW binding/wash-
ing buffer.
3. Allow the strip to equilibrate for 15 min on ice. Meanwhile, thaw an aliquot of
the 2.5-mg/mL DNase I stock solution.
4. Bring the tube to room temperature, place it in a scintillation vial, and determine
cpm of the probe retained on the strip by Cerenkov counting.
5. Transfer an equal amount of radioactivity of free probe to a separate, siliconized
0.5-mL Eppendorf tube containing 200 µL of SW binding/washing buffer, and
subject it to the same manipulation as its membrane-associated counterpart (step 3).
6. Prepare an appropriate dilution of DNase I in ice-cold distilled, deionized water.
Mix thoroughly by inversion and gentle vortexing.
7. Add to both samples 200 µL of DNase I reaction buffer and mix by flicking. Let
the tubes stand for 1 min at room temperature. It is not necessary to close the caps
on the tubes until addition of the DNase stop solutions (step 10).
8. Add dilute DNase I to a final concentration of 25 ng/mL and quickly distribute by
flicking. It is helpful—especially when footprinting several strips—to have all
necessary items (pipetmen, buffers, timer, DNase I) in close proximity. The
Combined SW–Nuclease Footprinting 145
smoother this procedure goes, the better (and more reproducible!) the footprint(s)
will be.
9. Incubate for 1 min at room temperature (see Note 9). As with solution footprinting
protocols, the exposure time for the free-DNA control reaction can be titrated to
achieve the cutting intensity profile of the membrane-bound form. Nevertheless,
we have found (employing probes of various lengths) that the above combination
of digestion time and DNase I concentration generates sufficient cleavage for a
good signal-to-noise ratio.
10. Following treatment, remove the strip (with forceps) from the tube and rapidly
immerse it in 500 µL of ice-cold DNase-STOP solution A. Leave on ice for 2 min
(do not vortex!). Terminate the control reaction (free probe) by adding 200 µL of
ice-cold DNase-STOP solution B; vortex thoroughly, spin briefly, transfer into a
siliconized 1.5-mL Eppendorf tube, and proceed directly to step 15.
11. Place the strip (using forceps) in a siliconized 0.5-mL Eppendorf tube containing
200 µL of probe elution buffer. Spin briefly in a microcentrifuge to submerge the
entire strip.
12. Incubate the tube on a vortexing shaker at 37°C for 2 h.
13. Add 100 µL of distilled, deionized water and vortex the tube vigorously (2 min).
14. Microcentrifuge for 2 min to pellet the NC strip, and transfer the supernatant into
a siliconized 1.5-mL Eppendorf tube. A Geiger counter can be used to monitor
efficient recovery of radioactivity; typically, >90% of the membrane-bound probe
is released by this process.
15. Extract once with phenol/chloroform/isoamyl alcohol (25:24:1; v/v) and once
with chloroform/isoamyl alcohol (24:1; v/v). In both cases, mix by vortexing (for
10 s) and spin for 5 min.
16. Transfer the aqueous (top) layer to a fresh siliconized 1.5-mL Eppendorf tube.
Precipitate the DNA with cold (–20°C) ethanol in the presence of 10 mM
MgCl
2
(added from the 0.5 M stock solution; MgCl
2
aids in the recovery of
small DNA fragments) and 10 µg carrier glycogen; mix by inversion, spin at
12,000g for 15 min.
17. Remove and discard the supernatant with a drawn-out Pasteur pipet, being care-
ful not to aspirate the DNA (the bottom of the tube can be held to a Geiger counter
to check that the DNA pellet remains). Add 800 µL of ice-cold 80% ethanol and
rinse the pellet by gently rolling the microcentrifuge tube. Respin for 3 min.
18. Remove and discard the supernatant using a drawn-out Pasteur pipet, taking
extreme care not to disturb the tiny, whitish pellet or the area of the tube where
the pellet should be located (the DNA pellet frequently adheres only loosely to
the walls of the tube). Dry the pellet in a SpeedVac rotary concentrator. Do not
allow the drying procedure to continue past the point of dryness because the
sample may be difficult to resuspend.
19. Dissolve the pellet in 6–8 µL of formamide loading buffer; pipet the loading
buffer onto the upper, inside surface of tube and tap the tube to drop the droplet
onto the DNA pellet. Vortex briefly at high speed for approx 15 s and micro-
centrifuge for 30 s to collect all of the solution to the bottom of the tube.
146 Papavassiliou
20. Transfer to fresh siliconized 1.5-mL Eppendorf tubes and determine the total
radioactivity recovered by Cerenkov counting each sample for 1 min in a scintil-
lation counter.
21. Heat samples to 95°C for 3 min to denature DNA and immediately chill in wet ice.
22. Spin briefly to bring the liquid to the bottom of the tubes. Samples can be electro-
phoresed immediately or stored at –70°C for no more than 24 h after footprinting.
23. Load 1500–2000 cpm of each DNA digestion product (adjust volumes accord-
ingly if necessary; it is essential that a consistent volume of sample be loaded on
each lane) onto a pre-electrophoresed, 5–10% denaturing urea (sequencing) gel
(see Notes 9b and 10a). Electrophorese in 1X TBE buffer at 60–70 W constant
power (for a 34 × 40 cm, 0.4-mm-thick gel) until the marker dye fronts have
migrated the appropriate distance in order to visualize the DNA region of interest
(11). To determine the location of the transcription factor-binding site(s), the
DNase I digests are electrophoresed alongside a Maxam–Gilbert G + A sequenc-
ing ladder prepared from the end-labeled footprint probe (14). See Chapter 7 for
a fast protocol for preparing such a ladder.
24. Disassemble gel apparatus, carefully lift off one glass plate and soak the gel (still
on the second glass plate) in fixing solution for 15 min (see Note 10b).
25. Drain briefly, overlay the gel with two sheets of 3MM paper, and carefully peel it
off the glass plate. Cover the gel surface with plastic wrap and dry under vacuum
at 80°C for approx 1 h.
26. Expose the dry gel to X-ray film overnight at –70°C with an intensifying screen
(a piece of paper placed between the gel and the film will prevent spurious expo-
sure of the film resulting from static electricity). Several different exposures may
be required to obtain suitable band densities.
27. Compare lanes corresponding to free and protein-bound DNAs to identify the
region(s) of protection; the region(s) of the DNA fragment that is bound by the
factor appears as a blank stretch (footprint) in the otherwise continuous back-
ground of digestion products.
4. Notes
1. The diversity of properties characteristic of DNA-binding transcription factors
imposes an empirical determination of the conditions under which the sample(s)
is prepared for SDS-PAGE. In some cases, the reducing agent (2-mercapto-
ethanol) should be omitted from the sample buffer and, in others, the SDS con-
centration should be lowered to 0.5%. Furthermore, some DNA-binding proteins
may not withstand the sample boiling before loading on the gel.
2. The presence of methanol in the Western transfer buffer may cause a problem
during the electrophoretic transfer of some bulky DNA-binding proteins (gel
shrinkage reduces pore size).
3. a. The commercially available nonfat dried milk preparations from various sup-
pliers contain large amounts of protein kinase/phosphatase activities; these
activities can potentially interfere with binding of the DNA probe to transcrip-
tion factors whose DNA-binding capacity is known, or suspected, to be subject to
Combined SW–Nuclease Footprinting 147
regulation by inducible phosphorylation/dephosphorylation events. If this is the
case, substitute nonfat dried milk in the SW blocking/renaturation buffer for
the recommended grade of lipid-free BSA; this particular grade contains only
trace amounts of the aforementioned activities and should be preferred as block-
ing agent (15,16). Moreover, the use of lipid-free BSA results in an even back-
ground throughout and enhances the specific signal-to-noise ratio in the
DNA-probing step (17).
b. By manipulating the conditions for renaturation of the membrane-immobilized
proteins (e.g., by incorporating a cycle of protein denaturation and renaturation
[16]), the method may be extended to the analysis of proteins resolved in two-
dimensional gels (18). This offers a powerful and convenient means for studying
cell cycle/type/stimulus-dependent DNA–transcription factor interactions and
their regulatory roles in gene activity.
4. a. Inclusion of poly(vinyl alcohol) (a molecular crowding agent [volume
excluder]) in the buffer decreases the amount of small ions/water available for
hydration of any probe dissociated from the immobilized protein matrix and ren-
ders the aqueous environment unfavorable for the unbound DNA. Consequently,
the effective concentration of the probe is increased and interactions with low
binding constants are stabilized.
b. It may be important in some cases to supplement this buffer with ZnSO
4
(Aldrich, Milwaukee, WI; final concentration 10 µM) if the transcription factor(s)
under study is known, or suspected, to contain a zinc-finger domain(s).
5. a. The DNA chosen for probing the protein blot can be a cis-acting regulatory (pro-
moter/enhancer) DNA restriction fragment in the size range of 125–250 bp, with the
putative transcription-factor-binding sites located no less than 20–25 bp from the
labeled end. This is to ensure that the region of DNA to be investigated for the pres-
ence of footprints is capable of being accurately resolved on a sequencing gel.
b. A prerequisite for the subsequent DNase I footprinting analysis is the use of a
DNA probe that has been labeled on only one strand of the DNA duplex. The
labeling of only one strand of a promoter/enhancer restriction fragment can be
achieved in a number of ways, such as using T
4
polynucleotide kinase and [γ-
32
P]
ATP (5'-end labeling), the large (Klenow) fragment of E. coli DNA polymerase I
and [α-
32
P] dNTPs (3'-end labeling [“filling in”]), or the polymerase chain reac-
tion (PCR) amplification (19). Preparation of radiolabeled DNA employing any
of these methodologies requires about 8 h. A combination of 5' and 3' end-labeled
DNA probes allows both strands to be analyzed side by side from the same end of
the DNA duplex.
c. For optimal sensitivity in the SW procedure, the probe should be of as high a
specific activity as possible and highly purified. The latter can be assured by
using a nucleic acid-specific, ion-exchange column, such as the Elutip™-d
(Schleicher & Schuell) or the NACS prepac cartridge (BRL, Gaithersburg, MD)
(11). It is recommended not to store the pure, labeled DNA probe longer than
2–4 d, because the radiation creates nicks in the DNA that will appear as addi-
tional bands in the sequencing gel.
148 Papavassiliou
6. Synthetic alternate copolymers, such as poly[dI–dC] · poly[dI–dC] or poly[dA–
dT] · poly[dA–dT] (Boehringer or Pharmacia), at similar final concentrations
may be more suitable competitors for some DNA-binding transcription factors.
7. Longer washing times at room temperature are detrimental resulting in dissocia-
tion of the bound probe, but longer washing times with cold (4°C) SW binding/
washing buffer can reduce background without significant signal loss. To reduce
possible low-specificity DNA–protein complexes, the final wash can be per-
formed in cold SW binding/washing buffer with higher salt concentration (i.e.,
100–200 mM KCl).
8. If prestained nonradioactive protein MW standards have been used, the edges of
the plastic wrap should be marked with pieces of tape labeled with radioactive
(or fluorescent) ink (let the ink dots dry completely before exposing to X-ray
film!). These marks allow the autoradiogram to be aligned with the protein size
markers on the NC sheet (Subheading 3.2., step 1), facilitating calculations on
the relative molecular weight of the specific DNA-bound protein species
(obtained radioactive signal[s]). If [
14
C]-methylated protein MW markers have
been used, their position will be apparent on the X-ray film without the need to
use the radioactive (or fluorescent) ink procedure.
9. a. Although longer digestion times do not enhance background cutting
(uncomplexed DNA is minimized), they may lead to substantial deviations from
the required “single-hit kinetics” (i.e., on average, each DNA molecule is nicked
at most once; this corresponds to nicking approx 30–50% of the DNA molecules).
b. Intense bands due to uncleaved, full-length probe should be visible at the top
of each lane in the DNA sequencing gel. This aids in determining whether single-
hit kinetics are operative and whether equal amounts of total radioactivity are
loaded in each lane.
10. a. If the DNA probe is relatively long (i.e., >175 bp) and multiple transcription-fac-
tor-binding sites are to be resolved, a gradient or wedge sequencing gel can be used.
b. Wedge-shaped gels must be soaked in fixing solution, followed by 5% glyc-
erol for 10 min prior to drying.
References
1. Galas, D. J. and Schmitz, A. (1978) DNase footprinting: A simple method for the
detection of protein-DNA binding specificities. Nucleic Acids Res. 5, 3157–3170.
2. Tullius, T. D. and Dombroski, B. A. (1986) Hydroxyl radical “footprinting”: high-
resolution information about DNA–protein contacts and application to λ repres-
sor and Cro protein. Proc. Natl. Acad. Sci. USA 83, 5469–5473.
3. Kuwabara, M. D. and Sigman, D. S. (1987) Footprinting DNA–protein complexes in
situ following gel retardation assays using 1,10-phenanthroline–copper ion: Escheri-
chia coli RNA polymerase–lac promoter complexes. Biochemistry 26, 7234–7238.
4. Johnsrud, L. (1978) Contacts between Escherichia coli RNA polymerase and a
lac operon promoter. Proc. Natl. Acad. Sci. USA 75, 5314–5318.
5. Garner, M. M. and Revzin, A. (1981) A gel electrophoresis method for quantify-
ing the binding of proteins to specific DNA regions: application to compo-