Moss Tom. DNA-protein interactions: principles and protocols
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EMSAs for Analysis of DNA–Protein 25
limiting amounts of small animal tissues such as spleen, pancreas or prostate
are available. In these cases, short microprocedures adapted to prevent pro-
tease actions can also be performed (46). Once the crude extract has been
obtained, its quality must be evaluated. An informative way to test extracts is
to assess the DNA-binding ability of the ubiquitously expressed transcription
factor Sp1. We have found this transcription factor to be particularly sensi-
tive to proteases (47). Little or no Sp1 binding to its high-affinity binding site
(5'-GATCATATCTGCGGGGCGGGGCAGACACAG–3') (48) is usually
indicative of a poor quality nuclear extract. Although such an assay is clearly
invaluable when crude extracts are obtained from established tissue-culture cells,
caution must be observed when extracts are prepared from whole animal tissues
because not all organs express Sp1 at the same level (22,47,49).
3. The analysis of crude extracts prepared from whole animal tissues by EMSA is
somewhat restricted because of the numerous enzymatic activities, such as pro-
teases and deacetylases, these may contain. Degradation of nuclear proteins by
endogenous proteases can be prevented by the addition of protease inhibitors.
Whole animal tissue extracts are also often contaminated with highly active
endogenous phosphatases. Tissues such as liver, kidney, and bone have been
reported to be rich in these enzymes (50), some of which substantially decrease
the sensitivity of the EMSA by removing the [
32
P]-labeled phosphate from the
DNA probe. Although addition of phosphatase inhibitors, such as tetrasodium
pyrophosphate or sodium fluoride, to the reaction buffer can efficiently prevent
dephosphorylation, we have found that the same can also be achieved by simply
reducing either the temperature at which the binding reaction is normally per-
formed (30 min of incubation at 4°C) or the time allowed for the DNA–protein
interaction to occur (as low as 1 min of incubation at 22°C) (21). Alternatively,
probes labeled by fill-out of unpaired 5' termini using T
4
DNA polymerase or the
Klenow fragment of DNA polymerase I and an appropriate [α-
32
P] dNTP may be
used. (See also Note 13.)
4. When double-stranded oligonucleotides are selected as labeled probes in
EMSA, we recommend their size be in the range 20–70 bp. When working
with subcloned DNA sequences, optimal signal strength and resolution can
be achieved using fragments of 50–250 bp. Although larger fragments may
be used, they require longer migration times in order to efficiently resolve
the potential DNA–protein complexes. Furthermore, larger labeled probes
are likely to bind an increased number of nuclear proteins, which may compli-
cate the interpretation of the results.
5. Handling [γ
-32
P] ATP requires that special care be taken when labeling the DNA
probes used in EMSA. The reader is referred to the standard procedures and the
guidelines on manipulation of radioactive materials in effect at each research
facility. Alternative procedures for nonradioactive probe labeling have been
reported for EMSA analyzes (32,33).
6. Acrylamide is a potent neurotoxic compound that is easily absorbed through skin.
Wearing gloves and a mask to avoid direct contact with the skin or inhalation is
26 Laniel, Béliveau, and Guérin
therefore required when manipulating dry acrylamide or acrylamide solutions.
Similar care should also be taken with polyacrylamide gels, as they may still
contain low levels of unpolymerized acrylamide. Acrylamide solution is light
sensitive and should be kept away from direct light. It is worth noting that
acrylamide and bis-acrylamide are slowly converted to acrylic and bisacrylic acid,
respectively, upon prolonged storage. To avoid the use of acrylamide, alternative
nontoxic gel matrices are available, whose resolution properties are comparable
to those of polyacrylamide (34,35). The use of agarose gels containing a non-
toxic synergistic gelling and sieving agent (Synergel™) that helps improve the
resolution of DNA–protein complexes has also been reported recently (36).
7. The concentration of the polyacrylamide gel used in EMSA is primarily dictated
by both the size of the labeled probe selected and the resolution of the DNA–
protein complexes obtained. It can vary from 4% with large labeled DNA frag-
ments (of over 150 bp in length) to 12% with synthetic oligonucleotides. Two (or
more) closely migrating DNA–protein complexes that would normally appear as
a single diffuse, smeary complex on a 4% gel can usually be resolved on a 8%
gel. However, although increasing the gel concentration usually improved the
resolution of DNA–protein complexes, other complexes became unstable in high
concentration gels.
8. Although we feel DNA–protein interactions are best revealed using the Tris–
glycine buffer system, some complexes may not be detectable under such condi-
tions. The alternative use of other running buffer systems with varying ionic
strength, such as Tris–acetate, pH 7.5 or TBE, pH 8.0 (23) is advisable in order to
explore a broader range of DNA–protein complexes.
9. Nonspecific DNA–protein interactions are usually prevented by the addition to
the reaction mix of 1–5 µg of a nonspecific competitor DNA. Although this is
clearly very effective when crude nuclear extracts are used, such high concentra-
tions of nonspecific competitor DNA were found to compete even for specific
DNA–protein complexes when enriched preparations of nuclear proteins are used
in EMSA (38). The more enriched the nuclear protein of interest, the lower the
amount of nonspecific competitor required. For example, we routinely use 1–2 µg
poly(dI:dC) with crude nuclear proteins, 250 ng when the nuclear extract is
enriched on a heparin–Sepharose column, and no more than 25–50 ng with puri-
fied or recombinant proteins.
10. The signal strength of a shifted DNA–protein complex can be substantially
increased by procedures which favor the interaction between the protein of interest
and its target sequence. This can easily be achieved with enriched preparations of
nuclear proteins either by increasing the amount of the labeled probe used or by
decreasing the concentration of poly(dI:dC), or both. Furthermore, the DNA-bind-
ing ability of some nuclear proteins proved to be highly dependent on the salt con-
centration (usually KCl) of the reaction mix. Transcription factors such as NF1-L
and Sp1 interact best with their respective target sequence in the presence of
100 mM and 150 mM KCl, respectively (20). It is therefore useful to evaluate the
optimum KCl concentration for complex formation on any given DNA probe.
EMSAs for Analysis of DNA–Protein 27
11. Ethidium bromide is a powerful carcinogen that also possesses a moderate toxic-
ity. Wearing gloves is essential when manipulating solutions that contain this
DNA dye. Decontamination of ethidium bromide-containing solutions can be
achieved using either hypophosphorous acid or potassium permanganate (see
ref. 41 for an overview and detailed protocols).
12. Nearly all vertical electrophoresis apparatus can be used to perform EMSA ana-
lyzes. Although gel electrophoresis is performed at room temperature in some
EMSA protocols, we recommend 4°C. With some apparatus this can be easily
achieved using a specially designed cooling unit. However, for apparatus not
equipped with a cooling unit, simply run the gel in a cold room.
13. Although 5' end-labeling of the selected DNA fragment is best done using poly-
nucleotide kinase, very efficient labeling can also be accomplished using alterna-
tive procedures, such as filling 5' protruding ends using the Klenow fragment of
E. coli DNA polymerase I (41), a particularly attractive alternative when crude
nuclear extracts rich in various phosphatases are used (in the event that no phos-
phatase inhibitors are used in the binding buffer). Larger DNA segments can also
be efficiently labeled by PCR.
14. Chemical synthesis of olignonucleotides yields a substantial proportion of interme-
diate products of progressively decreasing length. This is particularly true for larger
oligonucleotides, because the efficiency of each nucleotide addition normally
ranges between 98.5% and 99%. For a 40-mer oligonucleotide, this means that
60% of the synthesized products are of the correct length and that the remaining
40% range in size between 1 and 39 nucleotides. Further purification by high-
performance liquid chromatography, OPC column, or gel electrophoresis is rec-
ommended before annealing oligonucleotides. The loss of even a few bases at the
ends of the synthetic duplex may be sufficient to prevent protein binding and
therefore reduce the ability to detect the DNA–protein complex in EMSA.
15. We have found that the thickness of the native polyacrylamide gel strongly affects
the resolution of shifted DNA–protein complexes; the thinner the gel, the better
the resolution. We currently use 0.75-mm-thick gels.
16. Formation of DNA–protein complexes is highly dependent on the voltage
selected for their migration into the polyacrylamide gel (7). We have found that
reducing the migration time by running the EMSA at voltage higher than 120 V
or 8 V/cm (usually corresponding to 10 mA for a single gel) renders most DNA–
protein complexes unstable, preventing their detection.
References
1. 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-
nents of the Escherichia coli lactose operon regulatory system. Nucleic Acids
Res. 9, 3047–3059.
2. Fried, M. and Crothers, D. M. (1981) Equilibria and kinetics of lac repressor–
operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9,
6505–6525.
28 Laniel, Béliveau, and Guérin
3. Gerstle, J. T. and Fried, M. G. (1993) Measurement of binding kinetics using the
gel electrophoresis mobility shift assay. Electrophoresis 14, 725–731.
4. Fried, M. G. and Daugherty, M. A. (1998) Electrophoretic analysis of multiple
protein–DNA interactions. Electrophoresis 19, 1247–1253.
5. Cann, J. R. (1998) Theoretical studies on the mobility-shift assay of protein–DNA
complexes. Electrophoresis 19, 127–141.
6. Cann, J. R. (1989) Phenomenological theory of gel electrophoresis of protein-
nucleic acid complexes. J. Biol. Chem. 264, 17,032–17,040.
7. Vossen, K. M. and Fried, M. G. (1997) Sequestration stabilizes lac repressor-
DNA complexes during gel electrophoresis. Anal. Biochem. 245, 85–92.
8. Cann, J. R. (1997) Models of mobility-shift assay of complexes between dimeriz-
ing protein and DNA. Electrophoresis 18, 1092–1097.
9. Murakami, Y., Huberman, J. A., and Hurwitz, J. (1996) Identification, purifica-
tion, and molecular cloning of autonomously replicating sequence-binding pro-
tein 1 from fission yeast Schizosaccharomyces pombe. Proc. Natl. Acad. Sci. USA
93, 502–507.
10. Zhang, W., Shields, J. M., Sogawa, K., Fujii-Kuriyama, Y., and Yang, V. M.
(1998) The gut-enriched Kruppel-like factor suppresses the activity of the
CYP1A1 promoter in an Sp1-dependent fashion. J. Biol. Chem. 273, 17,917–17,925.
11. Tyree, C. M., George, C. P., Lira-DeVito, L. M., Wampler, S. L., Dahmus, M. E.,
Zawel, L., et al. (1993) Identification of a minimal set of proteins that is required for
accurate initiation of transcription by RNA polymerase II. Genes Dev. 7, 1254–1265.
12. Gille, J., Swerlick, R. A., and Caughman, S. W. (1997) Transforming growth fac-
tor-β-induced transcriptional activation of the vascular permeability factor (VPF/
VEGF) gene requires AP-2-dependent DNA binding and transactivation. EMBO
J. 16, 750–759.
13. Roy, A. L., Du, H., Gregor, P. D., Novina, C. D., Martinez, E., and Roeder, R. G.
(1997) Cloning of an Inr- and E-box-binding protein, TFII-I, that interacts physi-
cally and functionally with USF1. EMBO J. 16, 7091–7104.
14. Ng, J. Y. and Marians, K. J. (1996) The ordered assembly of the ØX174-type
primosome. I. Isolation and identification of intermediate protein–DNA com-
plexes. J. Biol. Chem. 271, 15642–15648.
15. Kironmai, K. M., Muniyappa, K., Friedman, D. B., Hollingsworth, N. M., and
Byers, B. (1998) DNA-binding activities of Hop1, a synaptonemal complex com-
ponent from Saccharomyces cerevisiae. Mol. Cell. Biol. 18, 1424–1435.
16. Wakasugi, M., and Sancar, A. (1998) Assembly, subunit composition, and foot-
print of human DNA repair excision nuclease. Proc. Natl. Acad. Sci. USA 95,
6669–6674.
17. Laniel, M. A., Bergeron, M. J., Poirier, G. G., and Guérin, S. L. (1997) A nuclear
factor other than Sp1 binds the GC-rich promoter of the gene encoding rat
poly(ADP-ribose) polymerase in vitro. Biochem. Cell Biol. 75, 427–434.
18. Eskild, W., Robidoux, S., Simard, J., Hansson, V., and Guérin, S. L. (1994) Bind-
ing of a member of the NF1 family of transcription factors to two distinct cis-
acting elements in the promoter and 5'-flanking region of the human cellular
retinol binding protein 1 gene. Mol. Endocrinol. 8, 732–745.
EMSAs for Analysis of DNA–Protein 29
19. Roy, R. and Guérin, S. L. (1994) The 30-kDa rat liver transcription factor nuclear
factor 1 binds the rat growth-hormone proximal silencer. Eur. J. Biochem. 219,
799–806.
20. Robidoux, S., Eskild, W., Kroepelin, C. F., Hansson, V., and Guérin, S. L. (1992)
Salt-dependent formation of DNA/protein complexes in vitro, as viewed by the
gel mobility shift assay. Biotechniques 13, 354–358.
21. Laniel, M. A. and Guérin, S. L. (1997) Improving sensitivity of the EMSA by
restricting tissue phosphatase activities. Biotechniques 24, 964–970.
22. Potvin, F., Roy, R. J., Poirier, G. G., and Guérin, S. L. (1993) The US-1 element
from the gene encoding rat poly(ADP-ribose) polymerase binds the transcription
factor Sp1. Eur. J. Biochem. 215, 73–80.
23. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A.,
et al. (1992) In Short Protocols in Molecular Biology, 2nd ed., Wiley, New York.
24. Bergeron, M. J., Leclerc, S., Laniel, M. A., Poirier, G. G., and Guérin, S. L. (1997)
Transcriptional regulation of the rat poly(ADP-ribose) polymerase gene by Sp1.
Eur. J. Biochem. 250, 342–353.
25. Granger-Schnarr, M., Lloubes, R., De Murcia, G., and Schnarr, M. (1988) Spe-
cific protein–DNA complexes: immunodetection of the protein component after
gel electrophoresis and Western blotting. Anal. Biochem. 174, 235–238.
26. Demczuk, S., Harbers, M., and Vennström, B. (1993) Identification and analysis
of all components of a gel retardation assay by combination with immunoblotting.
Proc. Natl. Acad. Sci. USA 90, 2574–2578.
27. Dyer, R. B. and Herzog, N. K. (1995) Immunodepletion EMSA: a novel
method to identify proteins in a protein–DNA complex. Nucleic Acids Res.
23, 3345–3346.
28. Yamamoto, H. (1997) DNA mobility shift assay coupled with SDS-PAGE for
detection of DNA-binding proteins. Biotechniques 22, 210–211.
29. Williams, M., Brys, A., Weiner, A. M., and Maizels, N. (1992) A rapid method
for determining the molecular weight of a protein bound to nucleic acid in a
mobility shift assay. Nucleic Acids Res. 20, 4935–4936.
30. Ossipow, V., Laemmli, U. K., and Schibler, U. (1993) A simple method to rena-
ture DNA-binding proteins separated by SDS-polyacrylamide gel electrophore-
sis. Nucleic Acids Res. 21, 6040–6041.
31. Wolf, S. S., Hopley, J. G., and Schweizer, M. (1994) The application of
33
P-label-
ing in the electrophoretic mobility shift assay. Biotechniques 16, 590–592.
32. Suske, G., Gross, B., and Beato, M. (1989) Non-radioactive method to visualize spe-
cific DNA–protein interactions in the band shift assay. Nucleic Acids Res. 17, 4405.
33. Ludwig, L. B., Hughes, B. J., and Schwartz, S. A. (1995) Biotinylated probes in
the electrophoretic mobility shift assay to examine specific dsDNA, ssDNA or
RNA–protein interactions. Nucleic Acids Res. 23, 3792–3793.
34. Ramanujam, P., Fogerty, S., Heiser, W., and Jolly, J. (1990) Fast gel electro-
phoresis to analyze DNA–protein interactions. Biotechniques 8, 556–563.
35. Vanek, P. G., Fabian, S. J., Fisher, C. L., Chirikjian, J. G., and Collier, G. B.
(1995) Alternative to polyacrylamide gels improves the electrophoretic mobility
shift assay. Biotechniques 18, 704–706.
30 Laniel, Béliveau, and Guérin
36. Chandrasekhar, S., Souba, W. W., and Abcouwer, S. F. (1998) Use of modified
agarose gel electrophoresis to resolve protein–DNA complexes for electrophoretic
mobility shift assay. Biotechniques 24, 216–218.
37. Revzin, A. (1989) Gel electrophoresis assays for DNA–protein interactions.
Biotechniques 7, 346–355.
38. Larouche, K., Bergeron, M. J., Leclerc, S., and Guérin, S. L. (1996) A careful use
of the non-specific synthetic competitor poly(dI–dC):poly(dI–dC) is advised
in DNA–protein interaction studies involving enriched nuclear proteins.
Biotechniques 20, 439–444.
39. Hassanain, H. H., Dai, W., and Gupta, S. L. (1993) Enhanced gel mobility shift
assay for DNA-binding factors. Anal. Biochem. 213, 162–167.
40. Zhang, X. Y., Asiedu, C. K., Supakar, P. C., and Ehrlich, M. (1992) Increasing the
activity of affinity-purified DNA-binding proteins by adding high concentrations
of nonspecific proteins. Anal. Biochem. 201, 366–374.
41. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) In Molecular Cloning, A
Laboratory Manual , 2nd ed. (Nolan, C., ed.), Cold Spring Harbor Laboratory,
Cold Spring Harbor, New York.
42. Harvey, M., Brisson, I., and Guérin, S. L. (1993) A simple apparatus for fast and
inexpensive recovery of DNA from polyacrylamide gels. Biotechniques 14, 942–948.
43. Larouche, K., Leclerc, S., Giasson, M., and Guérin, S. L. (1996) Multiple nuclear regu-
latory proteins bind a single cis-acting promoter element to control basal transcription of
the human α4 integrin gene in corneal epithelial cells. DNA Cell Biol. 15, 779–792.
44. Leclerc, S., Eskild, W., and Guérin, S. L. (1997) The rat growth hormone and
human cellular retinol binding protein 1 genes share homologous NF1-like bind-
ing sites that exert either positive or negative influences on gene expression in
vitro, DNA Cell Biol. 16, 951–967.
45. Graves, B. J., Johnson, P. F., and McKnight, S. L. (1986) Homologous recognition of
a promoter domain common to the MSV LTR and HSV Tk gene. Cell 44, 565–576.
46. Roy, R., Gosselin, P., and Guérin, S. L. (1991) A short protocol for micro-
purification of nuclear proteins from whole animal tissues. Biotechniques 11,
770–777.
47. Robidoux, S., Gosselin, P., Harvey, M., Leclerc, S., and Guérin, S. L. (1992)
Transcription of the mouse secretory protease inhibitor p12 gene is activated by
the developmentally regulated positive transcription factor Sp1. Mol. Cell. Biol.
12, 3796–3806.
48. Dynan,W. S. and Tjian, R. (1983) The promoter-specific transcription factor Sp1
binds to upstream sequences in the SV40 early promoter. Cell 35, 79–87.
49. Saffer, J. D., Jackson, S. P., and Annarella, M. B. (1991) Developmental expres-
sion of Sp1 in the mouse. Mol. Cell. Biol. 11, 2189–2199.
50. McComb, R. B., Bowers, G. N., and Posen, S. (1979) Alkaline Phosphatase.
Plenum, New York.
DNase I Footprinting 31
31
From:
Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed.
Edited by: T. Moss © Humana Press Inc., Totowa, NJ
3
DNase I Footprinting
Benoît Leblanc and Tom Moss
1. Introduction
DNase I footprinting was developed by Galas and Schmitz in 1978 as a
method to study the sequence-specific binding of proteins to DNA (1). In the
technique, a suitable uniquely end-labeled DNA fragment is allowed to inter-
act with a given DNA-binding protein and then the complex partially digested
with DNase I. The bound protein protects the region of the DNA with which it
interacts from attack by the DNase. Subsequent molecular-weight analysis of
the degraded DNA by electrophoresis and autoradiography identifies the region
of protection as a gap in the otherwise continuous background of digestion
products; for examples see Fig. 1. The technique can be used to determine the
site of interaction of most sequence-specific DNA-binding proteins but has
been most extensively applied to the study of transcription factors. Because the
DNase I molecule is relatively large as compared to other footprinting agents
(see Chapters 5 and 6 on the use of hydroxy radicals and diethylpyrocarbonate),
its attack on the DNA is relatively easily sterically hindered. Thus, DNase I
footprinting is the most likely of all the footprinting techniques to detect a
specific DNA–protein interaction. This is clearly demonstrated by our studies
on the transcription factor xUBF (see Fig. 1B). The xUBF interaction with the
Xenopus ribosomal DNA enhancer can be easily detected by DNase I foot-
printing but has still not been detected by other footprinting techniques.
DNase I footprinting can not only be used to study the DNA interactions of
purified proteins but also as an assay to identify proteins of interest within a
crude cellular or nuclear extract (e.g., see ref. 2). Thus, it can serve much the
same function as a gel shift analysis (EMSA, Chapter 2) in following a specific
DNA-binding activity through a series of purification steps. Because DNase I
footprinting can often be used for proteins that do not “gel shift” (UBF,
32 Leblanc and Moss
Fig. 1B), it has more general applicability. However, because of the need for a
protein excess and the visualization of the footprint by a partial DNA digestion
ladder, the technique requires considerably more material than would a gel
shift and cannot of itself distinguish individual components of heterogeneous
DNA–protein complexes.
DNase I (E.C. 3.1.4.5) is a protein approx 40 Å in diameter. It binds in the
minor groove of the DNA and cuts the phosphodiester backbone of both strands
Fig. 1. Examples of DNase I footprints. (A) Footprint (open box) of a chicken eryth-
rocyte DNA binding factor on the promoter of the H5 gene (2) (figure kindly donated
by A. Ruiz-Carrillo). (B) Interaction of the RNA polymerase I transcription xUBF
with the tandemly repeated 60 and 81b.p. Xenopus ribosomal gene enhancers. Both
(A) and (B) used 5' end-labeled fragments. Minus and plus refer to naked and
complexed DNA fragments, respectively, and G+A to the chemical sequence ladder.
DNase I Footprinting 33
independently (3). Its bulk helps to prevent it from cutting the DNA under and
around a bound protein. However, a bound protein will also usually have other
effects on the normal cleavage by DNase I, resulting in some sites becoming
hypersensitive to DNase I (see Figs. 1 and 2). It is also not so uncommon to
observe a change in the pattern of DNase cleavage without any obvious
extended protection (e.g., see Fig. 2).
Unfortunately, DNase I does not cleave the DNA indiscriminately, some
sequences being very rapidly attacked while others remain unscathed even after
Fig. 2. Course of digestion with increasing amounts of DNase I. Here xUBF was
footprinted on the Xenopus ribosomal promoter using a 5' end-labeled fragment. The
numbers above the tracks refer to the DNase I dilution (in units/µL) employed and
minus and plus to the naked and complexed DNAs, respectively. The predominant
footprints are indicated by open boxes.
34 Leblanc and Moss
extensive digestion (4). This results in a rather uneven “ladder” of digestion
products after electrophoresis, something which limits the resolution of the
technique, see naked DNA tracks in Figs. 1 and 2. However, when the protein-
protected and naked DNA ladders are run alongside each other, the footprints
are normally quite apparent. To localize the position of the footprints, G+A
and/or C+T chemical sequencing ladders of the same end-labeled DNA probe
(5) should accompany the naked and protected tracks (see Note 1). As a single
end-labeled fragment allows one to visualize interactions on one strand only of
the DNA, it is usual to repeat the experiment with the same fragment labeled
on the other strand. DNA fragments can be conveniently 5' labeled with T
4
polynucleotide kinase and 3' labeled using the Klenow or the T
4
DNA poly-
merases (fill out) or terminal transferase (e.g., see ref. 6). A combination of 5'
and 3' end labeling allows both DNA strands to be analyzed side by side from
the same end of the DNA duplex.
DNase I footprinting requires an excess of DNA-binding activity over DNA
fragment used. The higher the percent occupancy of a site on the DNA, the
clearer a footprint will be observed. It is therefore important not to titrate the
available proteins with too much DNA. This limitation can, in part, be over-
come when a protein also generates a gel shift. It is then feasible to fractionate
the partially DNase-digested protein–DNA complex by nondenaturing gel
electrophoresis and to excise the shifted band (which is then a homogeneous
protein–DNA complex) before analyzing the DNA by denaturing gel electro-
phoresis as in the standard footprint analysis (see Chapters 2, 4, 5, and 7 for
analogous procedures).
Footprinting crude or impure protein fractions usually requires that an excess
of a nonspecific competitor DNA be added. The competitor binds nonspecific
DNA-binding proteins as effectively as the specific labeled target DNA frag-
ment and hence, when present in sufficient excess, leaves the main part of the
labeled DNA available for the sequence specific protein. Homogeneous and
highly enriched protein fractions usually do not require the presence of a non-
specific competitor during footprinting. When planning a footprinting experi-
ment, it is a prerequisite to start by determining the optimal concentration of
DNase I to be used. This will be a linear function of the amount of nonspecific
DNA competitor but more importantly and less reproducibly, will be a func-
tion of the amount and purity of the protein fraction added. As a general rule,
more DNase is required if more protein is present in the binding reaction,
whether or not this protein binds specifically. Thus, very different DNase con-
centrations may be required to produce the required degree of digestion on
naked and protein-bound DNA. A careful titration of the DNase concentration
is therefore essential to optimize the detection of a footprint and can even make
the difference between the detection or lack of detection of a given interaction.