Moss Tom. DNA-protein interactions: principles and protocols
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Identification of DNA-Binding Proteins 255
255
From:
Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed.
Edited by: T. Moss © Humana Press Inc., Totowa, NJ
17
Identification of Sequence-Specific DNA-Binding
Proteins by Southwestern Blotting
Simon Labbé, Gale Stewart, Olivier LaRochelle, Guy G. Poirier,
and Carl Séguin
1. Introduction
Southwestern blotting was first described by Bowen et al. (1) and was used
to identify DNA-binding proteins that specifically interact with a chosen DNA
fragment in a sequence-specific manner. In this technique, mixtures of pro-
teins such as crude nuclear extracts or partially purified preparations are first
fractionated on a sodium dodecyl sulfate (SDS) denaturing gel; the gel is then
equilibrated in a SDS-free buffer to remove detergent and the proteins trans-
ferred by electroblotting to an immobilizing membrane. During the transfer the
proteins renature and hence DNA-binding proteins may subsequently be
detected on the membrane by their ability to bind radiolabeled DNA. Fraction-
ation of crude nuclear extracts on an SDS gel followed by electroblotting and
analysis for sequence-specific DNA binding directly on the blot combines
the advantages of a high-resolution fractionation step with the ability to rap-
idly analyze for a large number of different DNA-binding specificities.
The successful identification of specific DNA-binding proteins by the South-
western technique largely depends on the renaturation of the proteins after their
separation by SDS-polyacrylamide gel electrophoresis (PAGE). The ease with
which renaturation can be achieved after treatment with SDS varies from pro-
tein to protein. Some DNA-binding proteins may be inefficiently renatured
and thus be unable to bind DNA once they are adsorbed onto membranes. In
addition, any multimeric protein that requires a combination of different
molecular-weight subunits to bind DNA will be missed. Proteins requiring a
cofactor(s) in order to show their ability to specifically interact with DNA may
also be difficult to detect.
256 Labbé et al.
Site-specific protein–DNA interactions may also be obscured by the large
number of nonspecific DNA-binding proteins present in a typical crude nuclear
extract. Conditions for DNA binding, such as pH, ionic strength, and divalent
cation requirement, as well as the type and amount of nonspecific DNA used as
competitor, should be optimized for each protein under investigation. Special
attention should also be given DNA-binding proteins frequently present in
crude nuclear extracts and known to copurify with sequence-specific DNA-
binding proteins (see Note 1). Despite these shortcomings, Southwestern blot-
ting has, over the last few years, been used to identify and characterize several
sequence-specific DNA-binding proteins, histones, and nonhistone proteins;
for examples, see Fig. 1 and refs. 2–14, as well as RNA-binding proteins (15–17).
In addition, preparative Southwestern blots have been used to define DNA
sequences recognized by a given DNA-binding protein (18), whereas South-
western screening of cDNA libraries has been used to isolate several sequence-
specific DNA-binding proteins (19,20).
2. Materials
All solutions are made with double-distilled or Milli Q water.
1. Crude nuclear extracts or protein chromatographic fractions.
2. 1.5 M Tris-HCl, pH 8.8.
3. 0.5 M Tris-HCl, pH 6.8.
4. Sodium dodecyl sulfate (SDS) 10% and 2-bis-mercaptoethanol.
5. Tris–glycine buffer 10X: 250 mM Tris base and 1.92 M glycine pH 8.3. To make
this up, dissolve 30 g Tris base, and 144 g glycine in 1 L of water (see Note 2).
6. Electrophoresis running buffer: 2X Tris–glycine buffer containing 1% SDS.
7. Transfer buffer: Tris–glycine buffer 1X.
8. Binding buffer 1X: 20 mM HEPES, pH 7.9, 5 mM MgCl
2
, 50 mM NaCl, and 1 mM
dithiothreitol (DTT) (see Notes 3 and 4).
9. Sample loading buffer 2X: 125 mM Tris, pH 6.8, 4% (w/v) SDS, 20% (v/v) glyc-
erol, 10% (v/v) 2-bis-mercaptoethanol, and 0.025% (w/v) bromophenol blue.
Make 1-mL aliquots and store at –20°C.
10. Acrylamide (50:1) and (30:0.8):acrylamide:N',N'-methylene–bis-acrylamide
made up in water (see Note 5). Deionize the acrylamide solution with a mixed-
bed resin (21) and store at 4°C. Note: Acrylamide is a potent neurotoxin. Wear
gloves and a facemask when handling the dry powder.
11. 10% Ammonium persulfate (w/v) in water (prepared weekly and stored in
aliquots at –20°C ), and N,N,N',N'-tetramethylethylenediamine (TEMED).
12. Methanol and isobutanol.
13. Blocking buffer–5%: 5% (w/v) nonfat dried milk and 0.01% (v/v) Antifoam A
emulsion (Sigma) in 1X binding buffer.
14. Blocking buffer–0.25%: 0.25% (w/v) nonfat dry milk, 0.01% (v/v) Antifoam A
emulsion in 1X binding buffer.
Identification of DNA-Binding Proteins 257
Fig. 1. Southwestern analysis of mouse L-50 cell crude nuclear extracts. Approxi-
mately 200 µg of extracted protein was resolved by SDS-PAGE, transferred to a PVDF
membrane, and incubated with radiolabeled DNAs. Upper panel: Binding of labeled
oligos corresponding to control and mutant metal regulatory elements (MREs) of the
mouse metallothionein 1 gene promoter. Lanes: 1, control MREd; 2, Mutant-a; 3,
Mutant-b; 4, Mutant-c; 5, Mutant-d. Left lower panel: Analysis of the binding speci-
ficity of the MREd oligo to nuclear proteins using MREd as the probe and cold MREd
(lanes 1–3) or Mutant-a (lanes 4 and 5) as competitor. Right lower panel: Competi-
tion experiments using heterologous probe and nonspecific nucleic acids as competi-
tors. Probes: lanes, 1–3, MREd; lanes 4–6, a 309 bp pBR322 MspI fragment.
Nonspecific nucleic acid competitor cocktail was added as follows: lanes 1 and 4,
none; lanes 2 and 5, 0.8 µg/mL; lanes 3 and 6, 4 µg/mL. The arrow corresponds to M
r
108,000 and the arrowhead indicates the position of Mr 45,000. (From ref. 21.)
258 Labbé et al.
15. End-labeled oligonucleotides (oligos) or DNA fragments of high specific activ-
ity. Standard procedures (21) can be followed in the preparation of DNA samples
(see Note 6).
16. Poly(dA–dT)·poly(dA–dT), poly(dI–dC)·poly(dI–dC), calf thymus DNA, salmon
sperm DNA, Escherichia coli DNA.
17.
14
C-Labeled protein standards (5 µCi/mL, Amersham) and prestained protein
standards such as the Kaleidoscope Prestained Standards from Bio-Rad.
18. Vertical electrophoresis apparatus with a central cooling core (e.g., Protean II
slab cell, Bio-Rad) and glass plates (see Note 7).
19. Electroblotting apparatus with cooling coil (e.g., Trans-blot transfer cell, Bio-Rad).
20. Electrophoresis DC power supply capable of 200 V and 2 Amp (e.g., model 200/
2.0 of Bio-Rad).
21. Recirculating water chiller apparatus.
22. Immobilon™–PVDF membrane (Millipore).
23. Whatman 3MM paper.
3. Method
The Southwestern procedure is conducted in three stages: Crude nuclear
protein extracts or purified or partially purified protein preparations are sepa-
rated by electrophoresis on an SDS–polyacrylamide gel, transferred to the
PVDF membrane, and then the immobilized proteins assessed for their ability
to bind a labeled oligo corresponding to a given DNA cis-acting regulatory
element or a labeled DNA promoter fragment.
1. Prepare an 8% acrylamide–bis-acrylamide (50:1) separating gel (see Note 4).
Mix 25 mL 1.5 M Tris-HCl, pH 8.8, 15.7 mL acrylamide, and 57.2 mL water.
Filter the solution through a 0.45-µm membrane and then add 0.5 mL 10% SDS,
0.5 mL of 10% ammonium persulfate (APS), and 35 µL TEMED. Mix gently by
inversion and pour the gel. Gently overlayer the acrylamide solution with
isobutanol and allow the gel to set for at least 1 h. Remove the isobutanol, rinse
gel surface thoroughly with water. Prepare the stacking gel by mixing 6.25 mL of
0.5 M Tris-HCl, pH 6.8, 3.35 mL acrylamide–bis-acrylamide (30:0.8), 15 mL
water. Filter the solution and add 0.25 mL of 10% SDS, 0.25 mL of 10% APS and
17 µL TEMED. Mix gently, pour over the separating gel, add a comb, and allow
the stacking gel to set for at least 1 h. Just before using, carefully wash the wells
with running buffer using a syringe.
2. Dilute equal volumes of protein preparation and 2X loading buffer. We typically
use 100–400 µg (protein) of a crude nuclear extract (prepared according to
Dignam [22]) in the presence of a cocktail of four protease inhibitors (i.e.,
leupeptin, polymethylsulfonyl fluoride [PMSF], antipain, and chemostatin A).
Lower amounts are required when using purified or partially purified protein
preparations. Mix loading buffer and samples just before loading to avoid SDS
precipitation. We do not boil or otherwise purposely denature the protein prepa-
rations (see Note 8). Include 15 µL of
14
C-labeled protein markers in a separate
Identification of DNA-Binding Proteins 259
lane. Run gel in electrophoresis electrode buffer (2X Tris–glycine buffer, 1%
SDS) overnight at 75 V (constant voltage). Do not prerun the gel. We perform the
electrophoresis at 4°C by connecting the cooling coil provided with the electro-
phoresis apparatus to a recirculating water chiller (see Note 9).
3. Electrophoresis gels should be pre-equilibrated in transfer buffer prior to com-
mencement of electrophoretic transfer. Pre-equilibration will aid the removal of
SDS, contaminating the electrophoresis salts from the proteins, and will facilitate
subsequent renaturation to the native conformation (see Note 10). In addition,
the pretransfer step will allow any changes in the size of the gel resulting from
the swelling or shrinking to occur at this stage rather than during the transfer.
Thus, after the gel is run, dismantle the apparatus, remove the gel from the plates,
and soak it in transfer buffer for 60 min with gentle agitation. We usually per-
form this operation in the cold room (see Note 9). Wear clean disposable gloves.
4. Lay the gel onto 3MM paper prewetted with transfer buffer. Always keep the gel
wet by regularly pouring buffer over it.
5. Prewet the PVDF membrane in methanol for few seconds and transfer it to water.
6. Begin mounting the protein-blotting sandwich by placing one sponge (provided
with the blotting transfer apparatus) into the holder, followed by the 3MM support
paper and placing the gel (see Fig. 2 and Note 11).
7. Lay the prewetted PVDF membrane over the gel.
8. Carefully remove any air bubbles trapped between the gel and the membrane. We
use a 10-mL glass pipet gently rolled over the membrane.
9. Place a second prewetted (with transfer buffer) 3MM paper and repeat step 8.
Place the second foam pad onto the sandwich and close the holder. Introduce the
holder into the transfer apparatus, previously filled with cold transfer buffer. Be
sure that the membrane is toward the positive electrode. Place the transfer appa-
ratus on the stirring plate and connect it to a circulating cooler unit (see Note 12).
10. It is important that a stirring bar be placed inside the transfer apparatus and that
the transfer buffer be stirred during the course of the experiment. This will
help to maintain uniform conductivity and temperature during electrophoretic
Fig. 2. Schematic representation of the protein blotting sandwich assemblage used
in Southwestern experiments. The orientation of the sandwich in relation to the cath-
ode (+) and the anode (–) is indicated.
260 Labbé et al.
transfer. Failure to do so will result in poor transfer of proteins and may pose a
safety hazard.
11. Transfer at 50 V constant for 3 h.
12. After the transfer is completed, dismantle the apparatus, remove the membrane
from the gel, and successively place it in (1) 200 mL of binding buffer in a plastic
dish for 15 min with gentle agitation, (2) 250 mL blocking buffer–5% for a mini-
mum of 1 h with agitation, (3) and 150 mL blocking buffer–0.25% containing
10
6
cpm/mL of probe DNA and leave overnight with agitation (see Note 13).
Never let the membrane dry between the transfer and the binding steps.
13. After the transfer, the gel may be transferred in 50% methanol for subsequent
silver staining to visualize untransferred proteins (see Note 14).
14. Wash the membrane by successively soaking it under continuous agitation in
(1) blocking buffer–0.25% for 30 s, (2) blocking buffer–0.25% for 20 min, (3)
blocking buffer–0.25% for 20 min, and (4) Binding buffer 1X for 20 min. Recover
wash liquids because they contain
32
P.
15. Let the membrane dry on 3MM paper at room temperature for 1 h, then autorad-
iograph using an X-ray film and intensifying screen if necessary.
4. Notes
1. Crude nuclear extracts prepared from human HeLa and mouse L cells contain
three common nonspecific DNA-binding proteins that often contaminate
preparations of affinity-purified factors. These are poly (ADP–ribose) poly-
merase (PARP), which has an M
r
of 116,000 (as well as a typical proteolytic
fragment of M
r
-60,000) (27), the Ku antigen, which consist of two
polypeptides of M
r
-70,000 and 80,000 (28), and replication protein A (RP-A)
of M
r
-74,000 (29). The heat-shock protein hsp70 (M
r
-70,000), which can
stick rather tenaciously to proteins that are improperly folded, may also cause
problems. Thus, when Southwestern analyses are performed with affinity-
purified nuclear proteins, it is recommended to be suspicious of positive
polypeptides of M
r
-60,000, 70,000–74,000, 80,000, and 110,000, and to per-
form control experiments. For instance, in the case of PARP, if a polypeptide
with an M
r
in the range of this protein is detected in the Southwestern proce-
dure, a Western analysis could be performed on an aliquot of the protein
preparation using anti-PARP antibodies available commercially (Roche Diag-
nostics/Boehringer). Alternatively, a chromatographic step with Red Agarose
(Bio-Rad), which binds PARP with high affinity, can be performed to remove
of this possible contaminant (27). One should, however, bear in mind that
PARP has recently been shown to act as a coactivator for a number of tran-
scription factors, including AP-2 (30), E47 (31), and TEF-1 (32); thus, its
detection using a specific DNA probe corresponding to the binding site of a
known transcription factor could actually be functionally relevant.
2. Tris–glycine buffers should not be pH adjusted.
3. Dithiothreitol should be stored at –20°C, thawed on ice, and added when the
solution is ice-cold.
Identification of DNA-Binding Proteins 261
4. Several DNA-binding proteins require zinc ions to efficiently bind DNA. As a
precaution, we routinely add 5 µM ZnCl
2
to all solutions (loading, electrophore-
sis, transfer, binding, and wash buffers).
5. The percentage of gel and the ratio of acrylamide to bis-acrylamide will depend
on the M
r
of the protein of interest. We have used a ratio of 50:1 to increase
resolution of the high-molecular-weight protein species. A ratio of 30:0.8 or 29:1
may be more appropriate for average protein species. Plates are cleaned with
phosphate-free soap and rinsed with deionized water, Milli Q water, and ethanol
95%. We usually do not silanize the plates with dichlorodimethylsilane, as we
observed that it could interfere with the subsequent steps.
6. Synthetic oligos or DNA fragments can be conveniently 5' labeled with T
4
kinase
(21). A radiolabeled probe with a high specific activity increases the sensitivity
of detection and reduces background (23). A nonradioactive Southwestern pro-
cedure, using chemiluminescent detection, has also been described (24).
7. The procedure can both be carried out using a standard electrophoresis and trans-
fer apparatus or a small-size apparatus such as the Mini-protean II and mini-
Trans-Blot transfer cell of Bio-Rad (2).
8. We do not boil samples before electrophoresis in order to avoid irreversibly
denaturing proteins or to affect the DNA-binding properties of more sensitive
protein species (4).
9. Depending of the biochemical properties of the protein under investigation and
of its stability at room temperature, it is generally recommended to perform the
whole procedure at 4°C. Particular conditions of temperature may need to be
determined for an optimal binding of different factors to DNA.
10. Although the extent of renaturation is not known, this wash, by removing SDS
from the proteins, permits functional binding to the DNA recognition sequence.
Triton X-100 (1,12), urea (3,5,8,11) or guanidium HCl (9,10) can be included in
the transfer buffer to promote the removal of SDS and to facilitate subsequent
renaturation of the proteins. Low-molecular-weight proteins (≤10 kDa) may dif-
fuse more readily out of gels during the pre-equilibration step. To avoid this, one
can shorten the pre-equilibration period, changing the buffer several times to aid
equilibration. Methanol (20%) can also be added to the transfer buffer (5,9,25).
However, this organic solvent may affect the DNA-binding properties of the pro-
teins. The choice of renaturation procedure and buffers should be determined
empirically and will depend on the factors studied.
11. Each layer of the sandwich is thoroughly prewetted with buffer and then care-
fully positioned on top of the previous layer, taking care to avoid trapping any air
bubbles which would distort the resulting transfer.
12. Placing the trans-blot apparatus in the cold room is an inadequate means of con-
trolling transfer buffer temperature. Efficient heat removal is obtained by con-
necting the transfer apparatus to a recirculating cooler. Transfer is a function of
molecular weight, with the largest proteins being transferred more slowly. The
precise transfer conditions should take into account the molecular-weight range
of the proteins under investigation and the composition of the gel.
262 Labbé et al.
13. It is important to block any remaining free binding sites on the membrane in
order to reduce nonspecific binding of the DNA probe. The binding buffer should,
thus, be designed to optimize the specific binding of the radiolabeled probe while
keeping nonspecific binding to a minimum. Nonspecific binding can be reduced
by the addition of an excess of unlabeled DNA such as poly(dI–dC)·poly(dI–dC)
or poly(dA–dT)·poly(dA–dT), or sheared E. coli, salmon sperm, or calf thymus
DNA. Nonspecific binding can also be reduced by increasing salt concentrations
in the binding reaction and/or in the wash buffer. In addition, the use of lipid-free
bovine serum albumin (BSA) instead of nonfat dry milk has been reported to
optimize the signal-to-noise ratio (26). Although specific high-affinity DNA–
protein interactions will not normally be competed by nonspecific competitor
DNA, the addition of a large excess of competitor DNA may eventually do so.
Thus, the amount of competitor DNA and the salt concentration of buffers will
depend on the factor of interest. The specificity and affinity of binding should be
further examined by adding an unlabeled target sequence or a nonfunctional
mutated target sequence DNA as a competitor to the blocking and/or binding
solutions. Alternatively, to determine the specificity of an interaction, the bind-
ing of a radiolabeled mutant target DNA can be compared with the binding of the
unmutated sequence (see Fig. 1) (2).
14. Some proteins may not retain their ability to bind selectively with their target
DNA after immobilization of the protein on the membrane. Furthermore, the
membrane will not necessarily retain all of the proteins that have migrated out
the gel. It is, thus, useful to determine which proteins have been transferred to the
membrane. Detection of the transferred proteins may be achieved after autorad-
iography by staining the membrane with either Coomassie brilliant blue R-250,
Amido black, or Ponceau rouge. Proteins remaining in the gel after transfer can
be visualized by silver staining.
Acknowledgments
We thank R. Hancock for comments on the manuscript and S. Desnoyers for
helpful suggestions. S.L. is the recipient of a scholarship from FRSQ, and O.L.
of a Studentship from NSERC. This work was supported by a Grant from
NSERC to C.S.
References
1. Bowen, B., Steinberg, J., Laemmli, U. K., and Weintraub, H. (1980) The detec-
tion of DNA-binding proteins by protein blotting. Nucleic Acids Res. 8, 1–20.
2. Séguin, C. and Prévost, J. (1988) Detection of a nuclear protein that interacts with
a metal regulatory element of the mouse metallothionein 1 gene. Nucleic Acids
Res. 16, 10,547–10,560.
3. Jack, R. S., Brown, M. T., and Gehring, W. J. (1983) Protein blotting as a means
to detect sequence-specific DNA-binding proteins. Cold Spring Harb. Symp.
Quant. Biol. 47(Pt 1), 483–491.
Identification of DNA-Binding Proteins 263
4. Miskimins, W. K., Roberts, M. P., McClelland, A., and Ruddle, F. H. (1985) Use
of a protein-blotting procedure and a specific DNA probe to identify nuclear pro-
teins that recognize the promoter region of the transferrin receptor gene. Proc.
Natl. Acad. Sci. USA 82, 6741–6744.
5. Silva, C. M., Tully, D. B., Petch, L. A., Jewell, C. M., and Cidlowski, J. A. (1987)
Application of a protein-blotting procedure to the study of human glucocorticoid
receptor interactions with DNA. Proc. Natl. Acad. Sci. USA 84, 1744–1748.
6. Hughes, E. N., Engelsberg, B. N., and Billings, P. C. (1992) Purification of nuclear
proteins that bind to cisplatin-damaged DNA. Identity with high mobility group
proteins 1 and 2. J. Biol. Chem. 267, 13,520–13,527.
7. Wegenka, U. M., Buschmann, J., Lutticken, C., Heinrich, P. C., and Horn, F.
(1993) Acute-phase response factor, a nuclear factor binding to acute-phase
response elements, is rapidly activated by interleukin-6 at the posttranslational
level. Mol. Cell. Biol. 13, 276–288.
8. Kwast-Welfeld, J., Debelle, I., Walker, P. R., Whitfield, J. F., and Sikorska, M.
(1993) Identification of a new cAMP response element-binding factor by south-
western blotting. J. Biol. Chem. 268, 19,581–19,585.
9. Dhawan, P., Chang, R., and Mehta, K. D. (1997) Identification of essential nucle-
otides of the FP1 element responsible for enhancement of low density lipoprotein
receptor gene transcription. Nucleic Acids Res. 25, 4132–4138.
10. Villafuerte, B. C., Zhao, W., Herington, A. C., Saffery, R., and Phillips, L. S.
(1997) Identification of an insulin-responsive element in the rat insulin-like
growth factor-binding protein-3 gene. J. Biol. Chem. 272, 5024–5030.
11. Wu, J., Jiang, Q., Chen, X., Wu, X. H., and Chan, J. S. (1998) Identification of a
novel mouse hepatic 52 kDa protein that interacts with the cAMP response ele-
ment of the rat angiotensinogen gene. Biochem. J. 329, 623–629.
12. Melkonyan, H., Hofmann, H. A., Nacken, W., Sorg, C., and Klempt, M. (1998)
The gene encoding the myeloid-related protein 14 (MRP14), a calcium-binding
protein expressed in granulocytes and monocytes, contains a potent enhancer ele-
ment in the first intron. J. Biol. Chem. 273, 27,026–27,032.
13. Carrion, A. M., Mellstrom, B., and Naranjo, J. R. (1998) Protein kinase A-depen-
dent derepression of the human prodynorphin gene via differential binding to an
intragenic silencer element. Mol. Cell. Biol. 18, 6921–6929.
14. Chen, A. and Davis, B. H. (1999) UV irradiation activates JNK and increases alphaI(I)
collagen gene expression in rat hepatic stellate cells. J. Biol. Chem. 274, 158–164.
15. Katsu, Y., Yamashita, M., and Nagahama, Y. (1997) Isolation and characteriza-
tion of goldfish Y box protein, a germ-cell- specific RNA-binding protein. Eur. J.
Biochem. 249, 854–861.
16. Johnston, K. A., Polymenis, M., Wang, S., Branda, J., and Schmidt, E. V. (1998)
Novel regulatory factors interacting with the promoter of the gene encoding the
mRNA cap binding protein (eIF4E) and their function in growth regulation. Mol.
Cell. Biol. 18, 5621–5633.
17. Hamann, S. and Stratling, W. H. (1998) Specific binding of Drosophila nuclear
protein PEP (protein on ecdysone puffs) to hsp70 DNA and RNA. Nucleic Acids
Res. 26, 4108–4115.
264 Labbé et al.
18. Keller, A. D. and Maniatis, T. (1991) Selection of sequences recognized by a
DNA binding protein using a preparative Southwestern blot. Nucleic Acids Res.
19, 4675–4680.
19. Singh, H., Clerc, R. G., and LeBowitz, J. H. (1989) Molecular cloning of
sequence-specific DNA binding proteins using recognition site probes.
BioTechniques 7, 252–261.
20. Stuempfle, K. J. and Floros, J. (1997) Caution is advised when cDNA expression
libraries are screened by southwestern methodologies. BioTechniques 22, 260–264.
21. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Labo-
ratory Manual (Ford, N., Nolan, C., and Ferguson, M., eds.), Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY.
22. Dignam, J. D. (1990) Preparation of extracts from higher eukaryotes. Methods
Enzymol. 182, 194–203.
23. Handen, J. S. and Rosenberg, H. F. (1997) An improved method for southwestern
blotting. Front. Biosci. 2, c9–c11.
24. Dooley, S., Welter, C., and Blin, N. (1992) Nonradioactive Southwestern analysis
using chemiluminescent detection. BioTechniques 13, 540–543.
25. Schaufele, F., Cassill, J. A., West, B. L., and Reudelhuber, T. (1990) Resolution
by diagonal gel mobility shift assays of multisubunit complexes binding to a func-
tionally important element of the rat growth hormone gene promoter. J. Biol.
Chem. 265, 14,592–14,598.
26. Papavassiliou, A. G. and Bohmann, D. (1992) Optimization of the signal-to-noise
ratio in south-western assays by using lipid-free BSA as blocking reagent. Nucleic
Acids Res. 20, 4365–4366.
27. Mazen, A., Ménissier-de Murcia, J., Molinete, M., Simonin, F., Gradwohl, G.,
Poirier, G., et al. (1989) Poly(ADP-ribose) polymerase: a novel finger protein.
Nucleic Acids Res. 12, 4689–4698.
28. Mimori, T., Hardin, J. A., and Steitz, J. A. (1986) Characterization of the DNA-
binding protein antigen Ku recognized by autoantibodies from patients with rheu-
matic disorders. J. Biol. Chem. 261, 2274–2278.
29. Wold, M. S. (1997) Replication protein A: A heterotrimeric, single-stranded
DNA- binding protein required for eukaryotic DNA metabolism. Annu. Rev.
Biochem. 66, 61–92.
30. Kannan, P., Yu, Y., Wankhade, S., and Tainsky, M. A. (1999) PolyADP-ribose
polymerase is a coactivator for AP-2-mediated transcriptional activation . Nucleic
Acids Res. 27, 866–874.
31. Dear, T. N., Hainzl, T., Follo, M., Nehls, M., Wilmore, H., Matena, K., and
Boehm, T. (1997) Identification of interaction partners for the basic-helix-loop-
helix protein E47. Oncogene 14, 891–898.
32. Butler, A. J. and Ordahl, C. P. (1999) Poly(ADP-ribose) polymerase binds with
transcription enhancer factor 1 to MCAT1 elements to regulate muscle-specific
transcription. Mol. Cell. Biol. 19, 296–306.